Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof

ABSTRACT

Novel human monoclonal antibodies, and their corresponding nucleic acid sequences having high affinity for the human RSV-F protein are provided.

This application is a divisional of application Ser. No. 08/488,376,filed Jun. 7, 1995.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is a Parmixovirus of the Pneumovirusgenus which commonly infects the upper and lower respiratory tract. Itis so contagious that by age two, a large percentage of children havebeen infected by it. Moreover, by age four, virtually all humans have animmunity to RSV.

Typically, RSV infections are mild, remaining localized in the upperrespiratory tract and causing symptoms similar to a common cold whichrequire no extensive treatment. However, in some subjects, e.g.,immunosuppressed individuals such as infants, elderly persons orpatients with underlying cardiopulmonary diseases, the virus maypenetrate to the lower respiratory tract requiring hospitalization andbreathing support. In some of these cases, RSV infection may causepermanent lung damage or even be life threatening. In the United Statesalone, RSV results in about 90,000 hospitalizations each year, andresults in about 4500 deaths.

RSV appears in two major strain subgroups, A and B, primarily based onserological differences associated with the attachment glycoprotein, G.The major surface glycoprotein, i.e., the 90 kD G protein, can differ upto 50% at the amino acid level between isolates Johnson et al, Proc.Natl. Acad. Sci. (1987), 84, 5625-5629. By contrast, a potentialtherapeutic target, the 70 kD fusion (F) protein, is highly conservedacross different RSV strains, about i.e., 89% on the amino acid levelJohnson et al, J. Gen. Virol.(1988), 69, 2623-2628, Johnson et al, J.Virol. (1987), 10, 3163-3166, P. L. Collins, Plenum Press, NY (1991),103-162. Moreover, it is known that antibodies elicited againstF-protein of a given type are cross-reactive with the other type.

The F-protein is a heterodimer, generated from a linear precursor,consisting of disulfide-linked fragments of 48 and 23 kD respectively,Walsh et al, J. Gen. Virol, (1985), 66, 401-415. Inhibition of syncytiaformation by polyclonal antibodies is associated with significantreaction to the 23 kD fragment.

As noted, while RSV infections are usually mild, in some individuals RSVinfections may be life threatening. Currently, severe RSV infection istreated by administration of the antiviral agent Ribavarin. However,while Ribavarin exhibits some efficacy in controlling RSV infection, itsuse is disfavored for several reasons. For example, it is highlyexpensive and may be administered only in hospitals. Other known RSVtreatments only treat the symptoms of RSV infection and include the useof aerosolized bronchodilators in patients with bronchiolitis andcorticosteroid therapy in patients with bronchiolitis and RSV pneumonia.

To date, RSV vaccines intended to boost antiviral protective antibodieshave been largely unsuccessfil. For example, a vaccine based onformalin-inactivated RSV that was tested approximately 25 years ago,induced antibodies that were deficient in fusion inhibiting activityMurphy et al, Clinical Microbiology (1988), 26, 1595-1597, and sometimeseven exacerbated the disease. This may potentially be explained to theinability of the fornalin inactivated virus to induce protectiveantibodies. While high antibody titers were measured in vaccinerecipients, specific protective titers were lower than in the controlpopulation. This may be because formalin inactivated RSV does notdisplay the necessary conformational epitopes required to elicitprotective antibodies.

While there is no known effective RSV vaccine to date, there exists someclinical evidence that antibody therapy may confer protection againstRSV infection in susceptible individuals, and may even clear an existingRSV infection. For example, it has been reported that newborn infantsshow a low incidence of severe bronchiolitis, which is hypothesized tobe attributable to the presence of protective maternal antibodiesOgilvie et al, J. Med Virol (1981), 7, 263-271. Also, children who areimnune to reinfection exhibit statistically higher anti-F-protein titersthan those who are reinfected. Moreover, intravenous immune globulin(IVIG) prepared from high titer RSV-immune donors reduces nasal RSVshedding and improves oxygenation Hemming et al, Anti. Viral Agents andChemotherapy (1987), 31, 1882-1886. Also, recent studies have suggestedthat the virus can be fought and lung damage prevented by administeringRSV-enriched immune globulin (RSVIG) Groothuis et al, The New England J.Med. (1993), 329, 1524-1530, K. McIntosh, The New England J. Med.(1993), 329, 1572-1573, J. R. Groothuis. Antiviral Research, (1994), 23,1-10, Siber et al, J. Infectious Diseases (1994), 169, 1368-1373, Siberet al, J. Infectious Diseases (1992), 165:456-463.

Similarly, some animal studies suggest that antibody therapy with virusneutraling antibodies may confer protection against RSV or even clear anexisting RSV infection. For example, in vitro neutralizing mousemonoclonal antibodies have been reported to protect mice againstinfection and also to clear established RSV infections Taylor et al,J.Immunology, (1984), 52, 137-142, Stott et al, Immune Responses, VirusInfections and Disease, I.R.L. Press, London (1989), 85-104. Also,monoclonal antibodies to the F-protein of RSV have shown high efficacyin both in vitro and in vivo RSV models Tempest et al, Bio/Technology,(1991), 9, 266-271, Crowe et al, Proc. Natl. Acad. Sci. (1994), 91,1386-1390, Walsh et al, Infection and Immunity, (1984), 43, 756-758,Barbas III, et al, Proc. Natl. Acad. Sci. (1992), 89, 10164-10168,Walsh, et al, J. Gen. Virol. (1986), 67, 505-513. Antibodyconcentrations as low as 520-2000 μg/ki body weight have been reportedto result in almost instant recovery in animal studies Crowe et al,Proc. Natl. Acad. Sci. (1994), 91, 1386-1390. Moreover, these monoclonalantibodies have been disclosed to neutralize both A and B strains,including laboratory stains and wildtype strains. These antibodies wereadministered either by injection Groothuis et al. The New England J.Med. (1993), 329, 1524-1530, Siber et al. J. Infectious Diseases (1994),169, 1368-1373 or by aerosol Crowe et al, Proc. Natl. Acad. Sci. (1994),91, 1386-1390.

Two different types of potentially therapeutic monoclonal antibodies tothe RSV F-protein have been previously described in the literature,humanized murine antibodies Tempest et al, Biol. Technology, (1991) 9,266-271, or true human antibodies (Fab fragments) Barbas III. et al,Proc. Natl. Acad. Sci. (1992), 89, 10164-10168. Humanized murineantibodies were generated by CDR grafting a cross-strain neutralizingmurine anti-F-protein antibody onto a generic human Fc, as well asstructural areas of the variable part. The human Fab fragments wereproduced by combinatorial library technology using human bone marrowcells obtained from an HIV positive donor (immunocompromised). Thetherapeutic in vivo titers of the humanized and human RSV antibodieswere 5 and 2 mg/kg body weight, respectively. It is noted, however, thatthe hulmanized antibodies were tested in a syncytia inhibition assay,whereas the human anti-RSV Fab fragments were assayed to determine theirvirus neutralization activity. Therefore, the results reported with thehumanized and human anti-RSV antibodies are not directly comparable.

The Fab fragment generated by the combinatorial library technology weredisclosed to be efficient in aerosol. This is probably because of therelatively small size of the molecule. These results are highlyencouraging because a major target population for an RSV vaccine isinfants. Therefore, aerosol is a particularly desirable mode ofadministration.

However, notwithstanding the previous published reports of humanized andFab fragments specific to RSV, there still exists a significant need forimproved anti-RSV antibodies having improved therapeutic potential, inparticular anti-RSV antibodies which possess high affinity andspecificity for the RSV F-protein which effectively neutralize andprevent RSV infection.

Antibody therapy can be subdivided into two principally differentactivities: (i) passive immunotherapy using intact non-labeledantibodies or labeled antiodies and (ii) active immunotherapy usinganti-idiotypes for reestablishment of network balance in autoimmunity.

In passive immunotherapy, naked antibodies are administered toneutralize an antigen or to direct effector functions to targetedmembrane associated antigens. Neutralization would be of a lymphokine, ahormone, or an anaphylatoxin, i.e., C5a. Effector functions includecomplement fixation, macrophage activation and recruitment, and antibodydependent cell mediated cytotoxicity (ADCC). Naked antibodies have beenused to treat leukemia Ritz et al, S. F. Blood, (1981), 58, 141-152 andantibodies to GD2 have been used in treatments of neuroblastomas Schulzet al, Cancer Res. (1984), 44:5914 and melanomas Irie et al., Proc.Natl. Acad. Sci., (1986, 83:8694. Also, intravenous immune gammaglobulin (IVIG) antibodies with high anti-RSV titers recently were usedin experimental trials to treat respiratory distress caused by RSVinfection Hemming et al. Anti. Viral Agents and Chemotherapy, (1987),31, 1882-1886, Groothuis et al., The New England J. Med. (1993), 329,1524-1530, K. McIntosh, The New England J. Med. (1993), 329, 1572-1573,J. R. Groothuis, Antiviral Research, (1994), 23, 1-10, Siber et al. J.Infectious, Diseases (1994), 169, 1368-1373.

The therapeutic efficacy of a monoclonal antibody depends on factorsincluding, e.g., the amount, reactivity, specificity and class of theantibody bound to the antigen. Also, the in vivo half-life of theantibody is a significant therapeutic factor.

Still another factor which may significantly affect the therapeuticpotential of antibodies is their species of origin. Currently,monoclonal antibodies used for immunotherapy are almost exclusively ofrodent origin Schulz et al, Cancer Res. (1984), 44:5914, Miller et al,Blood (1981), 58, 78-86, Lanzavecchia et al, J. Edp. Med. (1988), 167,345-352, Sikora et al. Br. Med. Bull. (1984), 40:240, Tsujisaki et al,Cancer Research (1991), 51:2599, largely because the generation ofrodent monoclonal antibodies uses well characterized and highlyefficient techniques Kohler et al, Nature, (1975), 256:495, Galfre etal. Nature, (1977), 266:550. However, while rodent monoclonal antibodiespossess therapeutic efficacy, they can present restrictions anddisadvantages relative to human antibodies. For example, they ofteninduce sub-optimal stimulation of host effector functions (CDCC, ADCC,etc.). Also, murine antibodies may induce human anti-murine antibody(HAMA) responses Schroff et al, Can. Res. (1985, 45:879-885, Shawler etal, J. Immunol. (1985), 135:1530-1535. This may result in shortenedantibody half-life Dillman et al. Mod. (1986), 5, 73-84, Miller et al,Blood, (1983), 62:988-995 and in some instances may cause toxic sideeffects such as serum sickness and anaphylaxis.

In some subjects, e.g., heavily immunosuppressed subjects (e.g.,patients subjected to heavy chemical or radiation mediated cancertherapy Irie et al, Proc. Natl. Acad. Sci. (1986), 83:8694, Dillman etal, Mod. (1986), 5, 73-84, Koprowski et al. Proc. Natl. Acad. Sci.(1984), 81:216-219), use of murine monoclonal antibodies causes limitednegative side effects. By contrast, in patients with normal orhyperactive immune systems, murine antibodies, at least for some diseaseconditions may exhibit limited efficacy.

In an effort to obviate limitations of murine monoclonal antibodies,recombinant DNA techniques have been applied to produce chimericantibodies Morrison et al, Proc. Natl. Acad. Sci. (1984), 81:216219,Boulianne et al. Nature, (1984), 312, 644-646, humanized antibodies by"CDR grafting" Riechmann et al, Nature (1984), 332, 323-327 and"veneered" antibodies by substitution of specific surface residues withother amino acids to alleviate or eliminate antigenicity.

However, although such antibodies have been used successfully clinicallyGillis et al, J. Immunol. Meth (1989), 25:191, they have provencumbersome to produce. This is because the understanding of therequirements for optimal antigen recognition and affinity is not yetfully understood. Also, the human framework and the mouse CDR regionsoften interact sterically with a negative effect on antibody activity.Moreover, such antibodies sometimes still induce strong HAMA responsesin patients.

Human antibodies present major advantages over their murinecounterparts; they induce optional effector functions, they do notinduce HAMA responses and host antigen-specific antibodies may lead toidentification of epitopes of therapeutic value that may be too subtleto be recognized by a xenogeneic immune system Lennox et al, "MonoclonalAntibodies in Clinical Medicine." London: Academic Press (1982).

While human antibodies are highly desirable, their production iscomplicated by various factors including ethical considerations, and thefact that conventional methods for producing human antibodies are ofteninefficient. For example, human subjects cannot generally be adequatelyimmunized with most antigens because of ethical and safetyconsiderations. Consequently, reports of isolation of human monoclonalantibodies with useful affinities, ≧10⁸ molar to specific antigens arefew McCabe et al, Cancer Research, (1988), 48, 4348-4353. Also,isolation of anti-viral human monoclonal antibodies from donor primedcells has proved to be unwieldy. For example, Gorny reported that only 7of 14,329 EBV transformed cultures of peripheral blood mononuclear cells(PMBC's) from HIV positive donors resulted in stable, specific anti-HIVantibody producing cell lines Gorny et al, Proc. Natl. Acad. Sci.(1989), 86:1624-1628.

To date, most human anti-tumor antibodies have been generated fromperipheral blood lymphocytes (PBLs) Irie et al, Br. J. Cancer, (1981),44:262 or tumor draining lymph node lymphocytes Schlom et al, Proc. Nat.Acad. Sci. (1980), 77:6841-6845, Cote et al, Proc. Natl. Acad. Sci.(1983), 80:2026-2030 from cancer patients. However, such antibodiesoften react with intracellular, and thus therapeutically uselessantigens Ho et al, In Hybridoma Technology, Amsterdam (1988), 37-57 orare of the IgM class McCabe et al, Cancer Research (1988), 48,4348-4353, a class of antibodies with lesser ability to penetrate solidtumors than IgGs. Few of these human antibodies have moved to clinicaltrials Drobyski et al. R. C. Transplantation (1991), 51, 1190-1196,suggesting that the rescued antibodies may possess sub-optimalqualities. Moreover, since these approaches exploit the testing donorprimed B cells, it is clear that these cells are not an optimal sourcefor rescue of useful monoclonal antibodies.

Recently, generation of human antibodies from primed donors has been isimproved by stimulation with CD40 resulting in expansion of human Bcells Banchereau et al, F. Science (1991), 251:70, Zhang et al, J.Immunol. (1990), 144, 2955-2960, Tohma et al, J. Immunol. (1991),146:2544-2552 or by an extra in vitro booster step primer toimmortalization Chaudhuri et al, Cancer Supplement (1994), 73,1098-1104. This principle has been exploited to generate humanmonoclonal antibodies to Cytomegalovirus, Epstein-Barr Virus (EBV) andHemophilus influenza with cells from primed donors Steenbakkers et al.,Hum. Antibod. Hybridomas (1993), 4:166-173, Ferraro et al., HumanAntibod. Hybridomas (1993), 4:80-85, Kwekkeboom et al., ImmunologicalMethods (1993), 160:117-127, with a significantly higher yield thanobtained with other methods Gorny et al., Proc. Natl. Acad. Sci. (1989),86:1624-1628.

Moreover, to address the limitations of donor priming, immunization andcultivation ex vivo of lymphocytes from healthy donors has beenreported. Some success in generating human monoclonal antibodies usingex homine boosting of PBL cells from primed donors has been reportedMaeda et al, Hybridoma (1986), 5:33-41, Kozbor et al, J. Imnmunol.(1984), 14:23, Duchosal et al, Nature (1992, 355:258-262. Thefeasibility of immunizing in vitro was first demonstrated in 1967 byMishell and Dutton Mishell et al. J. Exp. Med (1967), 126:423-442 usingmurine lymphocytes. In 1973, Hoffman successfully immunized humanlymphocytes Hoffman et al. Nature (1973), 243:408-410. Also, successfulprimary immunizations have been reported with lymphocytes fromperipheral blood Luzzati et al, J. Exp. Med. (1975), 144:573:585, Misitiet al, J. Exp. Med. (1981), 154:1069-1084, Komatsu et al, Int. Archs.Allergy Appl. Immunol. (1986), 80:431-434, Ohlin et al, C. A. K.Immunology (1989), 68:325 (1989) tonsils Strike et al, J. Immunol.(1978), 132:1789-1803 and spleens, the latter obtained from trauma Ho etal, In Hybridoma Technology, Amsterdam (1988), 37-57, Boerner et al, J.Immunol. (1991), 147:86-95, Ho et al, J. Immunol. (1985), 135:3831-3838,Wasserman et al, J. Immunol. Meth. (1986), 93:275-283, Wasserman et al,J. Immunol. Meth. (1986), 93:275-283, Brams et al, Hum. Antibod.Hybridomas (1993), 4, 47-56, Brams et al, Hum. Antibod. Hybridomas(1993), 4, 57-65 and idiopathic thrombocytopenia purpura (ITP) patientsBoerner et al, J. Immunol. (1991), 147:86-95, Brams et al, Hum. Antibod.Hybridomas (1993) 4, 47-56, Brams et al, Hum. Antibod. Hybridomas(1993), 4, 57-65, McRoberts et al, "In Vitro Immunization in HybridomaTechnology". Elsevier, Amsterdam (1988), 267-275, Lu et al, P. Hybridoma(1993), 12, 381-389.

In vitro immunization offers considerable advantages, e.g., easilyreproducible immunizations, lends itself easily to manipulation ofantibody class by means of appropriate cultivation and manipulationtechniques Chaudhuri et al, Cancer Supplement (1994), 73, 1098-1104.Also, there is evidence that the in vivo tolerance to self-antigens isnot prevalent during IVI Boerner et al, J. Immunol. (1991), 147:86-95,Brams et al, J. Immunol. Methods (1987), 98:11. Therefore, thistechnique is potentially applicable for production of antibodies toself-antigens, e.g., tumor markers and receptors involved inautoimmunity.

Several groups have reported the generation of responses to a variety ofantigens challenged only in vitro, e.g., tumor associated antigens(TAAs) Boerner et al, J. Immunol. (1991), 147:86-95, Borrebaeck et al,Proc. Natl. Acad. Sci. (1988), 85:3995. However, unfortunately, theresulting antibodies were typically of the IgM and not the IgG subclassMcCabe et al. Cancer Research (1988), 48, 4348-4353, Koda et al, Hum.Antibod. Hybridomas, (1990), 1:15 and secondary (IgG) responses haveonly been reported with protocols using lymphocytes from immunizeddonors. Therefore, it would appear that these protocols only succeed ininducing a primary immune response but require donor immunized cells forgeneration of recall responses.

Also, research has been conducted to systematically analyze cultivationand immunization variables to develop a general protocol for effectivelyinducing human monoclonal antibodies in vitro Boerner, J. Immunol.(1991) 147:8695, Brams et al. Hum. Antibod. Hybridomas (1993), 4, 47-56,Lu et al, Hybridoma (1993), 12, 381-389. This has resulted in theisolation of human monoclonal antibodies specific for ferritin Boerneret al, J. Immunol. (1991), 147:86-95, induced by IVI of naive humanspleen cells. Also, this research has resulted in a protocol by which denovo secondary (IgG) responses may be induced entirely in vitro Brams etal, Hum. Antibod. Hybridomas (1993), 4, 57-65.

However, despite the great potential advantages of IVI, the efficiencyof such methods are severely restricted because of the fact that immunecells grow in monolayers in culture vessels. By contrast, in vivogerminal centers possessing a three-dimensional structure are found inthe spleen during the active phases of an immune response. Thesethree-dimensional structures comprise activated T- and B-cellssurrounded by antigen-presenting cells which are believed by themajority of immunologists to compare the site of antigen-specificactivation of B-cells.

An alternative to the natural splenic environment is to "recreate" ormimic splenic conditions in an immunocompromised animal host, such asthe "Severe Combined Immune Deficient" (SCID) mouse. Human lymphocytesare readily adopted by the SCID mouse (hu-SCID) and produce high levelsof immunoglobulins Mosier et al, Nature (1988), 335:256, McCune et al,L. Science (1988), 241, 1632-1639. Moreover, if the donor used forreconstitution has been exposed to a particular antigen, a strongsecondary response to the same antigen can be elicited in such mice. Forexample, Duchosal et al. Duchosal et al, Nature (1992), 355:258-262reported that human peripheral blood B-cells from a donor vaccinatedwith tetanus toxoid 17 years prior could be restimulated in the SCIDenvironment to produce high serum levels, i.e., around 10⁴. They furtherdisclosed cloning and expression of the genes of two human anti-TIantibodies using the lambda and the M13 phage combinatorial libraryapproach Huse et al. R. A. Science (1989), 246:1275 from the extactedhuman cells. The reported antigen affinities of the antibodies were inthe 10⁸ -10⁹ /M range. However, this protocol required donor primedcells and the yield was very low, only 2 clones were obtained from alibrary of 370,000 clones.

Therefore, previously the hu-SPL-SCID mouse has only been utilized forproducing human monoclonal antibodies to antigens wherein the donor haseither been efficiently primed naturally or by vaccination Stahli et al.Methods in Enzymology (1983), 92, 26-36, which in most cases involvesexposure to viral or bacterial antigens. Also, the reported serum titerlevels using the hu-SCID animal model are significantly lower than whatis typically achieved by immunization of normal mice.

Additionally, two protocols have been described by which induction ofprimary antibody responses can be followed by induction of secondaryantibody responses in hu-SCID mice using naive human lymphocytes.However, use of both of these protocols are substantially restricted. Inthe first protocol, primary responses are induced in hu-SCID mice intowhich human fetal liver, thymus and lymph nodes have been surgicallyimplanted. However, this method is severely restricted by the limitedavailability of fetal tissue, as well as the complicated surgicalmethodology of the protocol McCune et al, L. Science (1988), 241,1632-1639. In the second protocol, lethally irradiated normal mice werereconstituted with T- and B-cell depicted human bone marrow and SCIDmouse bone marrow cells Lubin et al, Science, (1991), 252:427. However,this method is disadvantageous because it requires a four monthincubation period. Moreover, both protocols result in very low antibodytiters, i.e., below 10⁴.

Also, Carlson et al. Carlsson et al. J. Immunol. (1992), 148:1065-1071described in 1992 an approach using PBMCs from an antigen (tetanustoxoid) primed donor. The cells were first depleted of macrophages andNK cells before being subjected to a brief in vitro cultivation andpriming period prior to transfer into a SCID mouse. The hu-SPL-SCIDmouse was then boosted with antigen. This method was reported to resultin average IT specific human IgG titers of ≈10⁴ in the hu-SPL-SCIDserum, with up to 5×10⁵ reported.

Production of human monoclonal antibodies further typically requires theproduction of immortalized B-cells, in order to obtain cells whichsecrete a constant, ideally permanent supply of the desired humanmonoclonal antibodies. Immortalization of B-cells is generally effectedby one of four approaches: (i) transformation with EBV, (ii) mouse-humanheterofusion, (iii) EBV traisformation followed by heterofasion, and(iv) combinatorial immunoglobulin gene library techniques.

EBV transformation has been used successfully in a number of reports,mainly for the generation of anti-HIV antibodies Gorny et al, Proc.Natl. Acad. Sci. (1989), 86:1624-1628, Posner, et al. J. Immunol.(1991), 146:4325-32. The main advantage is that approximately one ofevery 200 B-cells becomes transformed. However, EBV transformed cellsare typically unstable, produce low amounts of mainly IgM antibody,clone poorly and cease making antibody after several months ofculturing. Heterofusion Carrol. et al, J. Immunol. Meth. (1986),89:61-72 is typically favored for producing hybridomas which secretehigh levels of IgG antibody. Hybridomas are also easy to clone bylimiting dilution. However, a disadvantage is the poor yield, i.e., ≦1hybridomas per 20,000 lymphocytes Boerner. et al, J. Immunol. (1991),147:86-95, Ohlin. et al, C. A. K. Immunology (1989), 68:325, Xiu-mei etal. Hum. Antibod. Hybridomas (1990), 1:42, Borrebaeck C. A. K. Abstractat the "Second International Conference" on "Human Antibodies andHybridomas." Apr. 26-28, 1992, Cambridge, England. Combining EBVtransformation followed by heterofusion offers two advantages: (i) humanB-cells fuse more readily to the fusion partner after EBVtransformation, and (ii) result in more stable, higher producinghybridomas Ohlin. et al, Immunolozy (1989), 68:325, Xiu-mei. et al. Hum.Antibod. Hybridomas (1990), 1:42, Borrebaeck C. A. K. Absract at the"Second International Conference" on "Human Antibodies and Hybridomas."Apr. 26-28, 1992, Cambridge, England. The advantage of the finaltechnique, i.e., combinatorial immunoglobulin gene library technique isthe fact that very large libraries can be screened by means of the M13Fab expression technology Huse. et al, Science (1989), 246:1275, WilliamHuse, Antibody Engineering: A Practical Guide, Borrebaeck C. A. K., ed.5:103-120 and that the genes can easily be transferred to a productioncell line. However, the yield is typically extremely low, on the orderof 1 per 370,000 clones Duchosal, et al. Nature (1992), 355:258-262.

Thus, based on the foregoing, it is apparent that more efficient methodsfor producing hutman monoclonal antibodies, in particular antibodiesspecific to RSV, would be highly advantageous. Moreover, it is alsoapparent that human antibodies specific to the RSV F-protein havingsuperior binding affinity, specificity and effector functions than thosecurrently available would also be highly desirable.

OBJECTS OF THE INVENTION

It is an object of the invention to provide improved methods forproducing human antibodies of high titers which are specific to desiredantigens.

It is a more specific object of the invention to provide a novel methodfor producing high titer human antibodies which comprises (i) antigenpriming of naive human splenocytes in vitro, (ii) transferral of invitro antigen primed splenocyte cells to an immunocompromised donor,e.g., a SCID mouse, and (iii) boosting with antigen.

It is another specific object of the invention to provide improvedmethods for producing human monoclonal antibodies which are specific torespiratory syncytial virus (RSV), and in particular the RSV fusion (F)protein.

It is another object of the invention to provide an improved method forproducing EBV immortalized B-cels which favors the formation of EBVimmortalized B-cells which predominantly secrete IgG.

It is a more specific object of the invention to provide an improvedmethod for producing EBV immortalized human B-cells which predominandysecrete IgG's which comprises:

(i) antigen priming of naive human splenocytes in vitro;

(ii) transferal of such in vitro antigen primed naive splenocytes to animmunocompromised donor, e.g., a SCID mouse;

(iii) boosting the immunocompromised donor with antigen;

(iv) isolation of human antibody producing B-cells from the antigenboosted immunocompromised donor, e.g., SCID mouse; and

(v) EBV transformation of said isolated human antibody producingB-cells.

It is another object of the invention to provide novel compositionscontaining EBV transformed human B-cells obtained from SCID mice whichpredominandy secrete human IgG's.

It is a more specific object of the invention to provide novelcompositions containing EBV transformed human B-cells whichpredominantly secrete human IgG's produced by a method comprising:

(i) antigen priming of naive human splenocytes in vitro;

(ii) transferral of resulting in vitro antigen primed naive splenocytesto an immunocompromised animal donor, e.g., a SCID mouse;

(iii) boosting the immunocompromised animal donor, e.g., SCID mouse,with antigen;

(iv) isolation of human antibody producing B-cells from the antigenboosted immunocompromised donor, e.g., SCID mouse; and

(v) EBV transformation of said isolated human antibody producingB-cells.

It is another specific object of the invention to produce RSVneutralizing human monoclonal antibodies having an affinity (Kd) to theRSV F-protein of ≦2×10⁻⁹ Molar.

It is still another object of the invention to provide EBV immortalizedcell lines which secrete RSV neutralizing human IgG monoclonalantibodies having an affinity (Kd) to the RSV F antigen of ≦2×10⁻⁹Molar.

It is a more specific object of the present invention to provide two EBVimmortalized cell lines, RF-2 and RF-1, which respectively secrete humanmonoclonal antibodies also referred to as RF-2 and RF-1 which neutralizeRSV in vivo and each possess an affinity (Kd) for the RSV F-protein of≦2×10⁻⁹.

It is another object of the invention to transfect eukaryotic cells withDNA sequences encoding the RF-1 or RF-2 heavy and light variable domainsto produce transfectants which secrete human antibodies containing thevariable domain of RF-1 or RF-2.

It is a more specific object of the invention to provide transfected CHOcells which express the RF-1 or RF-2 heavy and light variable domains.

It is another object of the invention to treat or prevent RSV infectionin humans by administering a therapeutically or prophylacticallyeffective amount of RSV neutralizing human monoclonal antibodies whichare specific to the RSV F-protein and which exhibit a Kd for the RSVF-protein of ≦2×10⁻⁹ molar.

It is a more specific object of the invention to treat or prevent RSVinfection in humans by administering a therapeutically orprophylactically effective amount of RF-1 or RF-2 or a human monoclonalantibody expressed in a transfected eukaryotic cell which contains andexpresses the variable heavy and light domains of RF-1 or RF-2.

It is another object of the invention to provide vaccines for treatingor preventing RSV infection which comprise a therapeutically orprophylactically effective amount of human monoclonal antibodiesspecific to the RSV F-protein having a Kd for the RSV F-protein of≦2×10⁻⁹ molar, which neutralize RSV in vitro, in combination with apharmaceutically acceptable carrier or excipient.

It is a more specific object of the invention to provide vaccines fortreating or preventing RSV infection which comprise a therapeutically orprophylactically effective amount of RF-1 or RF-2 or human monoclonalantibodies derived from a transfected eukaryotic cell which contains andexpresses DNA sequences encoding the variable heavy and light domains ofRF-1 or RF-2, in combination with a pharmaceutically acceptable carrieror excipient.

It is another object of the present invention to provide a method fordiagnosis of RSV infection by assaying the presence of RSV in analytes,e.g., respiratory fluids using human monoclonal antibodies which possessan affinity (Kd) for the RSV fusion (F) protein or ≦2×10⁻⁹ molar.

It is still another object of the invention to provide novelimmunoprobes and test kits for detection of RSV infection which comprisehuman monoclonal antibodies specific to the RSV F-protein, which possessan affinity (Kd) for the RSV F protein of ≦2×10⁻⁹ molar, whichantibodies are directly or indirectly attached to a suitable reportermolecule, e.g., an enzyme or a radionuclide. In the preferred embodimentthese human monoclonal antibodies will comprise RF-1 or RF-2 orrecombinant human monoclonal antibodies produced in eukaryotic cells,e.g., CHO cells, which are transfected with the variable heavy and lightdomains of RF-1 or RF-2.

BRIEF DESCRIPTION OF THE INVENTION

The present invention in its broadest embodiments relates to novelmethods for making human antibodies to desired antigens, preferablyantigens involved in prophylaxis, treatment or detection of a humandisease condition. These methods comprise antigen priming of nativehuman splenocytes in vitro, transferral of the resultant in vitroantigen primed splenocyte cells to an immunocompromised donor, e.g., aSCID mouse, and boosting said immunocompromised donor with antigen.

The present invention also relates to methods for producing Epstein-BarrVirus (EBV) immortalized B-cells which favors the production of cellswhich secrete IgGs comprising: antigen priming of naive humansplenocytes in vitro; transferral of resultant in vitro antigen primedsplenocytes to an immunocompromised donor, e.g., a SCID mouse; boostingthe immunocompromised donor with antigen; isolating human antibodysecreting -cells, preferably IgG secreting, from the antigen boostedimmunocompromised donor, e.g., SCID mouse; and EBV transformation ofsaid isolated human antibody secreting cells.

The present invention more specifically relates to improved methods formaking human antibodies to RSV, in particular the RSV fusion (F) proteinwhich exhibit high affinity to RSV F-protein and which also neutralizeRSV infection, as well as the human monoclonal antibodies which resultfrom these methods. This is preferably effected by priming of naivehuman splenocytes in vitro with II-2 and optionally the RSV F-protein;transferral of the resultant in vitro primed splenocyte cells to animmunocompromised donor, e.g., a SCID mouse, and boosting with RSVF-protein to produce human B-cells which secrete neutralizing anti-RSVF-protein human antibodies having high affinity (Kd) to the RSVF-protein, i.e., ≦2×10⁻⁹ molar.

The resultant B-cells are preferably immortalized so as to provide aconstant stable supply of human anti-RSV F-protein monoclonalantibodies. In the preferred embodiment B-cells are isolated from theantigen boosted SCID mouse and transformed with EBV virus to produce EBVtransformed human B-cells which predominantly secrete human IgGs.

These cells are then cloned to select EBV transformed cell lines whichsecrete human monoclonal antibodies having high affinity (Kd) to RSVF-protein, i.e. ≦10⁻⁷ and preferably ≦2×10⁻⁹ molar.

The present invention also relates to the use of such anti-RSV F-proteinhuman monoclonal antibodies as therapeutic and/or prophylactic, as wellas diagnostic agents. As noted, the subject methods result in thegeneration of human monoclonal antibodies which exhibit high affinity(Kd) to the RSV F-protein, i.e., which possess a Kd for the RSVF-protein of ≦2×10⁻⁹ molar, which also neutralize RSV in vitro.Therefore, these antibodies are ideally suited as prophylactic andtherapeutic agents for preventing or treating RSV infection given thefact that the RSV F-protein is a surface protein which is highlyconserved across different RSV isolates. Also, given the high affinityand specificity of the subject human monoclonal antibodies to RSVF-protein, they also may be used to diagnose RSV infection.

More specifically, the present invention provides two particular humanmonoclonal antibodies to the RSV F-protein, i.e., RF-1 and RF-2, as wellas recombinant human antibodies derived therefrom, which are preferablyproduced in CHO cells, which cells have been transfected with DNAsequences encoding the variable heavy and light domains of RF-1 or RF-2.These antibodies are particularly useful as prophylactic and/ortherapeutic agents for treatment or prevention of RSV infection.Moreover, these antibodies are useful as diagnostic agents because theybind the RSV F-protein with high affinity (Kd), i.e., each possessaffinity for the RSV F-protein of ≦2×10⁻⁹. They are especially useful astherapeutic agents because of their high affinity and specificity forthe RSV F-protein, and their ability to effectively neutralize RSVinfection in vitro.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts immunoblot of F protein with anti-F protein hu-SPL-SCIDsera: Notice (A) and denatured (B) F protein was run in SDS-PAGE andtransferred to nitrocellulose by Western blot. Nitrocellulose stripswere reacted with positive control mouse anti-F protein MAb (Ganes 1Aand 1B), negative control hu-SPL-SCID serum anti-TT (lanes 2A and 2B)and hu-SPL-SCID anti-F protein sera from mice #6 (lanes 3A and 3B), #3(Ganes 4A and 4B) and #4 (lanes #5A and 5B).

FIG. 2 depicts immunofluorescence of HEP-2 cells with hu-SPL-SCID seraanti-F protein. Uninfected (left) and RSV-infected HEp-2 cells werereacted with serum from hu-SPL-SCID mouse #6 diluted 1:50 taken 15 daysafter boost. Binding was revealed GAH IGG-FITC.

FIG. 3 depicts the reactivity of purified RF-1 and RF-2 to plastic boundaffinity purified RSV F-protein. The reactivity of a reference humananti-RSV serum, LN, is also recorded. The ELISA plate was coated with 50ng RSV F-protein.

FIG. 4 depicts IEF of RF-1 (lane 2) and RF-2 Gane 3) human MAb purifiedfrom tumor cell supernatants. IEF was performed on a pH gradient of3-10. Lane 1 represents the pi standards.

FIG. 5 depicts indirect Immunofluorescence flow cytometry assay of HEp-2cells and HEp-2-cells infected with RSV, 1×10⁶, incubated with variousamounts of RF-1 and subsequently with a FITC-labeled GAH IgG. Therelative average intensity of the entire population is recorded.

FIG. 6 depicts NEOSPLA vector used for expression of human antibodies.CMV=cytomegalovirus promoter. BETA=mouse beta globin major promoter.BGH=bovine growth hormone polyadenylation signal. SVO=SV40 origin ofreplication. N1=Neomycin phosphotransferase exon 1. N2=Neomycinphosphotransferase exon 2. LIGHT=Human immunoglobulin kappa constantregion. Heavy=Human immunoglobulin gamma 1 or gamma 4 PE constantregion. L=leader. SV SV40 polyadenylation region.

FIG. 7a SEQ ID NO.:12! depicts the amino acid and nucleic acid sequenceof the variable light domain of RF-1.

FIG. 7b SEQ ID NO.:13! depicts the amino acid and nucleic acid sequenceof the variable heavy domain of RE-1.

FIG. 8a SEQ ID NO.:14! depicts the amino acid and nucleic acid sequenceof the variable light domain of RF-2.

FIG. 8b SEQ ID NO.:15! depicts the amino acid and nucleic acid sequenceof the variable heavy domain of RF-2.

FIG. 9a SEQ ID NO.:16! depicts the amino acid and nucleic acid sequenceof the RE-1 light chain, the leader sequence, and the human kappaconstant domain sequence.

FIG. 9b SEQ ID NO.:17! depicts the amino acid and nucleic acid sequenceof the RE-1 heavy chain, a leader sequence, and the human gamma/constantdomain sequence.

FIG. 9c depicts the human constant domain sequence.

FIG. 10 depicts schematically the NEOSPLA vector, referred to as NSKE1containing the RF-1 nucleic acid sequence and human gamma/constantdomain set forth in FIGS. 9a-9c.

FIG. 11a SEQ ID NO.:18! depicts the amino acid and nucleic acid sequenceof the RF-2 light chain, leader sequence, and human Kappa constantdomain.

FIG. 11b SEQ ID NO.:19! depicts the amino acid and nucleic acid sequenceof the RF-2 heavy chain, leader sequence, and human gamma/constantdomain:

FIG. 11c depicts the amino acid and nucleic acid sequence of the humangamma/constant domain.

FIG. 12 depicts schematically the NEOSPLA expression vector, referred toas NSKG1 containing the RF-2 nucleic acid sequences and humangamma/constant domain sequences set forth in FIG. 11a-11c.

DETAILED DESCRIPTION OF THE INVENTION

As discussed, the present invention provides a novel highly efficientmethod for producing human monoclonal antibodies to desired antigens,preferably antigens which are involved in a human disease condition.Antigens involved in a human disease condition typically will be surfaceantigens which comprise suitable therapeutic targets for antibodies. Forexample, this includes surface proteins of viruses and antigensexpressed on the surface of human cancer cells. In the preferredembodiment, the surface antigen will comprise the fusion protein(F-protein) of RSV.

Human disease conditions includes by way of example viral infections,e.g., RSV, papillomavirus, hepatitis, AIDS, etc., cancer, bacterialinfections, yeast infections, parasite infection, e.g, malaria, etc.Essentially, human disease conditions are intended to embrace any humandisease condition potentially preventable or treatable by theadministration of human monoclonal antibodies specific to a particularantigen.

The subject method for producing human monoclonal antibodies essentiallyinvolves the combination of in vitro priming of naive human spleencells, transferal of these spleen cells to immunocompromised donors,i.e., SCID mice, followed by antigen boosting of SCID mice which havebeen administered said spleen cells. It has been surprisingly discoveredthat the combination of these two known methods for producing humanantibodies results in synergistic results. Specifically, it results invery enhanced antigen specific responses to the immunizing antigen aswell as very high titers of human monoclonal antibodies of the IgGisotype. More specifically, it has been found that this combinationresults in unprecedented high secondary responses: the human IgGresponses in the hu-SPL-SCID serum were 10-fold higher than thoseresulting from transfer of naive cells in SCID and specific antibodyresponses were 1000-fold increased. Also, the resulting antibodies arefound to be of high affinity and specificity comparable to antibodiesproduced in experimentally hyperactive immune animals. It has also beenfound that when using naive spleen cells, to obtain such unexpectedresults it is necessary to challenge with antigen both in vitro andafter introduction into the resultant hu-SPL-SCID mouse. Also, it ispreferable but not essential to introduce additional fresh non-primedspleen cells to the hu-SPL-SCID donor just prior to antigen boosting.This has been found to result in still further enhancement of theantibody response.

The present invention was developed after an optimal in vitro primaryand boosting protocol for the generation of secondary responses fromnaive human spleen cells had previously been disclosed Brams et al. Hum.Antibod. Hybridomas (1993), 4, 57-65. The protocol Brams et al, Hum.Antibod. Hybridomas (1993), 4, 57-65 was found to provide for antigenspecific IgG responses about 2 to 10 times higher than obtained fromcultures subjected to one antigen challenge. This in vitro immunization(IVI) protocol was developed and optimized using very differentantigens, i.e., horse ferritin (HoF), calmodulin, prostate specificantigen (PSA), mouse IgG, transferrin, Keyhole Limpet Hemocyanine (KLH)di-nitro phenyl (DNP) bound to T-cell dependent protein carriers and RSVfusion (F) protein.

Essentially, this protocol involves restimulation of the spleen cellculture on day 1 after culturing is started with antigen together withautologous spleen cells in a 1:1 ratio. It has been demonstrated thatthe IgG responses measured using this protocol were the result ofrepeated antigen exposure, and are equivalent to secondary responses.

These experiments further demonstrated that intact spleens were theoptimal source of lymphocytes, including trauma- and ITP spleens. Bycontrast, peripheral blood lymphocytes (PBLs), and cells from tonsils orlymph nodes proved to be inferior for induction of antigen-specificresponses. Moreover, depletion or neutralization of any cellularcomponent resulted in inferior responses Boerner et al, J. Immunol.(1991), 147:86-95. Also, these experiments indicated that for a givenspleen cell preparation and antigen, that there exists a unique optimalantigen concentration.

Therefore, having established an optimal in vitro primary and boostingprotocol for generation of secondary responses from naive human spleencells; it was conceived to test this protocol in combination withprevious in vivo methods for producing human monoclonal antibodies,i.e., the SCID mouse. It was unknown prior to testing what effect, ifany, administration of antigen primed spleen cells would have on theresultant production of human monoclonal antibodies to a given antigenby the SCUD mouse or the ability of human lymphocytes to be maintainedtherein. However, it was hoped that this would provide for enhancedantigen boost and enhanced expression of the in vitro antigen primednaive spleen cells.

In this regard, it has been previously reported that human lymphocytescan establish themselves and remain alive for several months in SCIDsMcCune et al, Science (1988), 241, 1632-1639, Lubin et al, Science(1991), 252:427. However, as noted, surpa previous methods using SCIDsor human monoclonal antibodies to antigens have used cells from donorspreviously exposed to the antigen either naturally or by vaccination andhave typically not resulted in high human antibody titers.

Quite surprisingly, it was found that combination of in vitro primaryand boosting protocol for generation of secondary responses from humannaive spleen cells Brams et al, Hum. Antibod. Hybridomas (1993), 4,57-65 in the hu-SCID model resulted in synergistic results as evidencedby highly significant antigen specific IgG responses to the immunizingantigen.

Further, it was also discovered that the combination of these methods(using horse ferritin (HoF) as a model antigen) that:

(i) introduction of an in vitro immunization step prior to transfer intoSCIDs is essential for reliably inducing significant antigen-specificresponses;

(ii) human cells must be transferred into the peritoneum to achieveoptional maintenance of human splenocytes in the SCID mouse;

(iii) optimal in vitro cultivation is about three days;

(iv) use of IL-2 and optionally IL-4 or IL-6 in vitro results in highestantibody titers of antigen specific responses in the hu-SPL-SCID mice;

(v) the hu-SPL-SCID in mouse is preferably boosted with antigenemulsified in an adjuvant, e.g., Freunds Complete Adjuvant (FCA) and/orAlum;

(vi) killing or neutralization of NK cells, whether of murine or humanorigin surprisingly has no benefit on antibody production. However, itwas found that use of the SCID-beige mouse, an NK low line, as the hostfor the in vitro primed cells, provides for a superior response whenboosting is effected using a combination of adjuvants, i.e., FCA andAlum.

(vii) spleens, but not lymph nodes of≈1/3 of the hu-SPL-SCID mice wereenlarged up to 25 times compared to normal SCIDs. Moreover, of these upto two-thirds of the cells in such spleens tested positive for normalhuman lymphocyte membrane markers.

More specifically, the subject method comprises priming naive humansplenocytes in vitro, for about 1 to 10 days, preferably about 3 dayswith antigen, transferral of the primed cells to a SCID mouse, andsubsequently boosting the mouse with antigen about 3 to 14 days later,preferably about 7 days later. This has been demonstrated to result inhigh antigen specific IgG responses in the sera of the resultanthu-SPL-SCID mouse from about day 24 onwards. Typically, the serumend-dilution titers are about 10⁶ (half maximal responses atapproximately 50 mg IgG/ml) using a naive antigen, horse ferritin and107 (half maximal responses at approximately 5 mg IgGm/ml) when a recallresponse is induced with a viral antigen, i.e., the fusion protein ofRSV. It is expected based on these results that similar responses willbe obtained using other antigens.

As noted, optimal induction of the desired antibody response requiresantigen challenge of the human cells both in vitro and in vivo in thehu-SPL-SCID mouse. It was also found that IL-2 is necessary during invitro priming, and that IL-4 and IL-6 administered concomitantly withIL-2 further enhanced responses in the hu-SPL-SCID mouse. Moreover, SCIDreconstitution is facilitated but was not dependent on concomitantintraperitoneal administration of irradiated allergenic lymphocytes.

It was further discovered that there was significant variation in theantibody responses from one spleen to another. For example, some spleensrequired concomitant administration of antigen and fresh autologousspleen cells on day 10 for generation of antigen specific antibodyresponses. Also, it was found that the level of antibody responsesvaried somewhat in different hu-SPL-SCID mice. However, based on theteachings in this application, one skilled in the art can readily selectsuitable conditions so as to produce an optimal antigen specificantibody response to a given antigen.

For example, by testing several different spleen preparations for theirability to produce specific antibody in culture, e.g., after ten days ofin vitro immunization, one can identify the highest responder. Moreover,since large numbers of cells are prepared and frozen from each spleen,it is possible to set up a new in vitro immunization for three days fromthe selected spleen and follow up with transfer in SCID mice. Bycontrast, other cellular materials, e.g., peripheral blood cells are notamenable to such optimization, given the fairly limited amount of PBL'srecoverable from one donor in a single transferral.

As previously noted, in contrast to previous reports, it was found thatfor the present method, when peripheral blood cells were used,neutralization of human NK activity had no effect on spleens. Moreover,neutralization of SCID NK cells with complement fixing anti-asialo GMIantibodies decreased antigen-specific IgG responses. By contrast, use ofthe SCID/beige mouse, a strain with reduced NK cell levels did providefor significantly increased antigen specific IgG responses compared tonormal SCID.

Additionally, two immunization routes, intravenous (IV) andintraperitoneal (IP) were compared for their ability to provide forreconstitution of SCED mice, i.e., maintenance of spleen cells thereinand the production of human antibodies. It was found that the peritoneumwas the optimal site of cell transfer and immunization. Moreover, date,transfer of cells intravenously has never been found to result inrepopulation when more than 0.01 μg/ml human IgG was detected in themouse serum.

It was also found that the resultant IgG concentrations directlycorrelated with the number of transferred human cells. For example,repopulation of SCIDs was 92% when 5×10⁶ in vitro primed spleen cellswere injected intraperitoneally, and virtually 100% when 5×10⁷ in vitroprimed spleen cells were injected intraperitoneally. One skilled in theart can, based on the teachings in this application, select an optimalnumber of injected in vitro primed spleen cells. In general, this willrange from about 10⁴ to about 10⁸ cells, more preferably about 10⁶ to10⁸ cells, and most preferably at least about 10⁷ to 10⁸ cells.

It was also found that the antibody response is affected by the presenceof the particular adjuvant. More specifically, it was observed thatmaximal human antibody responses were achieved when the hu-SPL-SCID micewere boosted with antigen emulsified in Complete Freund's adjuvant (CFA)or using CFA and Alum together. Tests in hu-SPC-SCID boosted withferritin showed that CFA was a better adjuvant than Alum, eliciting 33mg and 13 mg/ml human IgG respectively. Combination of CFA and Alum didnot improve response in SCID. However, use of these adjuvants inSCID/beige-hu (which mice comprise a mutation resulting in reduced NKcell activity) results in 8-10 fold increase in IgG production comparedto CFA alone. However, it is expected that other adjuvants, orcombinations thereof, may also produce similar or even enhanced results.The highest total human IgG concentrate using Complete Freund's adjuvantand Alum together was about 10 mg/ml, and the specific highest IgGconcentration was about 500 μg/ml monoclonal antibody equivalent.

Using this method with ferritin produced polyclonal antibody responsescomparable to that obtained in hyperimmune goats, rabbits and pigs interms of specificity, reactivity, and use of Ig chain isotypes. Thehu-SPL-SCID serum antibodies were mostly IgG, bound only to cells fromtissues high in ferritin, and not to cells from ferritin-low orferritin-negative tissues, and recognized both natural ferritin as wellas denatured ferritin in a Western blot. These results are extremelyunexpected both in antibody concentration and the antigen specificity ofhuman antibodies obtained. Moreover, similar results are obtained usingdifferent antigens.

After injection, it is found that human cells tend to accumulate at twosites, i.e., the peritoneum and the mouse spleen. While no more thanabout 7% of human cells were found in the blood, the lymph nodes and theliver were of human antigen, between 25% and 33% of the cells were ofhuman origin in enlarged spleens and in the occasional tumors in someanimals. These human cells were almost exclusively B and T-cells, with asmall amount of CD14⁺ cells, mostly monocytes, in the enlarged spleens.

These results were determined by flow cytometry investigating spleen,lymph nodes, liver and peritoneum. In those cases that the humansplenocytes repopulated the spleen, it was found that the spleens wereoften enlarged, up to 25 times the size of native SCID spleens. Thehuman cells constituted up to about 30% of the total number of cells inthe spleen when measured immediately after extraction, with theremainder of unknown origin. However, after 3 days in culture, amajority of surviving cells were found to be of human origin as thecells bound antibodies and exhibited no cross reactivity with mouselymphocytes.

It was further observed that the reconstituted mice could be dividedinto two groups, those with normal size spleens and those with enlargedspleens. Hu-SPL-SCID mice with enlarged spleens, i.e., 25 times normalsize had human IgG levels approximately 150 times higher than those withnormal spleens, and the level of antigen specific human IgG wasapproximately 10,000 higher in those with normal size spleens which weretreated similarly. It was also found that the relative affinity of theantigen specific response increased throughout the response, indicatingthat a higher percentage of the total immunoglobulin pool was comprisedof antibodies having better binding properties. These results indicatethat the system is antigen driven.

These results are highly significant and indicate that it shouldgenerally be possible to rescue human cells from the hu-SPL-CID and usesame for generating combinatorial human antibody gene libraries therebyresulting in human monoclonal antibodies of high affinity andspecificity that may be used clinically and/or diagnostically.

More specifically, the present invention provides novel human monoclonalantibodies to the RSV F-protein which exhibit high affinity to the RSVF-protein, i.e., ≦2×10⁻⁹ molar protein and which human monoclonalantibodies are capable of neutralizing RSV in vitro. The presentinvention further provides methods for manufacture of such humanmonoclonal antibodies to the RSV F-protein.

In general, such human antibodies are produced by in vitro immunizationof naive human splenocytes with RSV F-protein, transferral of such invitro immunized human splenocytes into an immunocompromised animaldonor, i.e., a SCID mouse, boosting said animal with RSV F-protein, andisolation of human B cells therefrom which secrete human monoclonalantibodies to the RSV F-protein, immunization of said human B cells, andcloning of said immunilized B cells to select cells which secrete humanmonoclonal antibodies having a high affinity to RSV F-protein,preferably at least 10⁻⁷ molar and more preferably ≦2×10⁻⁹ molar.

As discussed, it has been discovered that the combination of in vitroimmunization, in particular of human splenocytes, i.e., which have orhave not been previously exposed to the RSV F-antigen and transferred toan immunocompromised animal donor, i.e., SCID mouse which is thenboosted with RSV F-protein antigen affords significant advantagesrelative to conventional methods for making human antibodies in SCIDmice. Namely, it provides for very high antibody titers, i.e., thehighest anti-F protein titers being about 10⁻⁷, high IgG concentrations,i.e., about 3 mg/ml for the highest responders. Moreover, this methodallows for the production of human antibodies having highly advantageouscombinations of properties, i.e., which exhibit both high affmity to theRSV F-protein and which moreover display substantial in vitroneutralizing activity.

As described in greater detail in the examples, the present inventorshave isolated two human monoclonal antibodies, RF-1 which exhibits anaffinity constant Ka to the F-protein, Ka=10¹⁰ M when determined byplasmin resonance, and RF-2 which exhibits an affmity constant ofKa=5×10⁸ M when determined by titration microcolorimetry. Also, thecalculated Kd of RF-2 was 2×10⁻⁹ M. Moreover, both of these antibodiesdisplay in vitro virus neutralizing properties at concentrations ofbetween 8 and 120 ng/ml as well as exhibiting an ability to inhibit thefusion of previously RSV infected cells. Significantly, this in vitroneutralization activity is applicable against a broad variety ofdifferent wild and laboratory RSV strains, both of the A and B virustypes.

Given these results, i.e. the high affinity of the subject antibodies tothe RSV F-protein, which comprises a surface protein expressed on thesurface of RSV infected cells, as well as ability to effectivelyneutralize the virus, and to inhibit fusion of virally infected cells,the subject human monoclonal antibodies should be suitable both astherapeutic and prophylactic agents, i.e., for treating or preventingRSV infection in susceptible or RSV infected subjects. As noted, RSVinfection is particularly prevalent in infants, as well as inimmunocompromised persons. Therefore, the subject monoclonal antibodieswill be particularly desirable for preventing or treating RSV infectionin such subjects.

Moreover, given the human origin of the subject monoclonal antibodies,they are particularly suitable for passive immunotherapy. This isbecause they likely will not be subject to the potential constraints ofmurine monoclonal antibodies, i.e., HAMA responses and absence of normalhuman effector functions. In fact, based on the characterization of thesubject human monoclonal antibodies (described in examples infra), itwould appear that both RF-1 and RF-2 exhibit substantially greater invitro neutralization activity and ability to inhibit fusion ofpreviously infected RSV cells than previously disclosed murine orchimeric anti-F protein antibodies and human Fab fragments derived fromrecombinational libraries. Also, given their human origin it is expectedthat such neutralization activity will be maintained upon in vivoadministration.

Another advantage of the subject human monoclonal antibodies is theirsubstantial absence of reactivity with normal tissues. As shown infra,the subject human monoclonal antibodies bind only to RSV infected cells,not to cell lines representing lymphoid tissue, liver, prostrate orlaryngeal epidermis. Therefore, these antibodies upon in vivoadministration should efficiently bind to RSV infected cells and not tonormal tissues and thereby should provide for neutralization of RSVinfection. Further, based on the disclosed properties, it is expectedthat the subject human monoclonal antibodies to the RSV F-protein may beused to protect susceptible hosts against RSV infection.

More specifically, the subject human monoclonal antibodies to the RSVF-protein are produced by obtaining human splenocytes, e.g., from atrauma or ITP source, which are then primed in vitro. This essentiallycomprises culturing said naive human splenocytes in vitro in thepresence of a sufficient amount of IL-2 and optionally RSV F-protein toinduce immunization, also referred to as antigen priming. In general,the amount of RSV F-protein that may be used ranges from about 1 to 200ng/ml RSV protein, more preferably 10 to 100 ng/ml, and most preferablyabout 40 ng/ml of RSV F-protein.

The in vitro culture medium will preferably also contain lymphokines, inparticular IL-2 and optionally IL-4 and IL-6. The amount thereof will beamounts which provide for immunization and the desired production ofantibody producing cells. For example, in the case of IL-2, an amountranging from about 5 to 200 IU/ml, and more preferably from about 10 to50 IU/ml, most preferably 25 IU/ml is suitable.

This culture medium will also contain other constituents necessary tomaintain the viability of human splenocytes in culture, e.g., aminoacids and serum. In the examples, a culture medium containing IMDMsupplemented with 2mM glutamine, 2 mM sodium pyruvate, non-essentialamino acids, 25 IU/ml IL-2 and 20% fetal calf serum was used. However,one skilled in the art, based on the teachings in this application, canvary the culture medium using routine optimization.

The in vitro immunization step will be effected for a time sufficient toinduce immunization In general, the cells will be cultured in thepresence of RSV F-protein from about 1 to 10 days and preferably forabout 3 days. However, this will vary dependent, e.g., upon theparticular spleen sample. Similarly, one skilled in the art, based onthe teachings in this application and using known methods may determinea suitable duration for the in vitro immunization step.

The antigen used for the in vitro immunization will preferably be apurified RSV F-protein so as to ensure that the splenocytes areimmunized against the F-protein and not against other useless(non-surface) antigens. Methods for obtaining purified RSV protein areknown in the art. The present inventors in particular utilized themethod of Walsh et al. J. Gen Virol., 70, 2953-2961, 1989. However, theparticular method is not critical provided that RSV F-protein ofsufficient purity to obtain human monoclonal antibodies havingspecificity to the RSV F-protein are obtained. Alternatively, the RSVF-protein may be produced by recombinant methods as described in U.S.Pat. No. 5,288,630 issued on Feb. 22, 1994.

After in vitro immunization, the RSV F-protein immunized or primed naivehuman splenocytes are then introduced into an immunocompromised donor,i.e., a SCID mouse. This is preferably effected by intraperitoneallyadministering the RSV F-protein primed human splenocytes into SCID mice.The number of such splenocytes which is administered will typically varyfrom about 10⁴ to 10⁸ spleen cells, with about 10⁷ to 10⁸ spleen cellsbeing preferred. The number of such cells is that which results in thedesired reconstitution, i.e., SCID mice which produce recoverableconcentrations of human antibodies specific to the RSV F-protein.Preferably, such spleen cells will be suspended in HBSS at aconcentration of about 8×10⁸ cells/ml prior to administration.

After intraperitoneal transferral of splenocytes, the SCID mice are thenboosted with the RSV F-protein. This is effected at a time sufficientlyproximate to the transferal of splenocytes such that the desiredproduction of human anti-RSV F-protein antibodies is realized. Ingeneral, this may be effected 3 to 14 days after transferral, andoptimally about 7 days after transferral. Preferably, said antigenadministration will be effected intraperitoneally. The amount of RSVF-protein administered will range from about 1 to 50 μg and preferablyabout 1 to 10 μg. In the examples, 5 μg protein was administered.However, the amount and time of immunization may vary dependent upon theparticular mouse, spleen sample, and purity of RSV F-protein.

Preferably, antigen boosting will be effected in the presence of anadjuvant, e.g., Complete Freund's Adjuvant, Alum, Saponin, etc., withComplete Freund's Adjuvant (CFA) and Alum being preferred. However, itis expected that other known adjuvants may be substituted to obtainsubstantially equivalent or even enhanced results.

After antigen boosting, the SCID mice are then bled, e.g., tail bled,and their serum tested for human IgG concentration and anti-F proteinantibody titers. Those animals which exhibit the highest antibody titersand concentration are then used for recovery of human IgG secretingcells.

It has been discovered that SCID mice having the highest anti-F humanantibody titers developed large abdominal tumors which provide a goodsource of human antibody secreting cells. Preferably, these tumors arerecovered by excision under sterile conditions, single cell suspensionsare prepared, and the cells are then washed and cultured. In theexamples, the cells are washed with IMDM containing 2% fetal calf serum,and the cells cultured in suspension of 10⁶ cells/ml in T-25 flaskscontaining IMDM with 10% FCS. However, such culturing conditions may bevaried by one skilled in the art.

These cells are then immortalized preferably using EBV. Immortalizedcells which secrete anti-F protein antibodies are then identified byknown methods, e.g., ELUSA. As noted, this method has been demonstratedto result in the identification of two distinct human monoclonalantibodies which specifically bind RSV F-protein, i.e. RF-1 and RF-2.However, based on the teachings in their application, in particular theexamples, other human monoclonal antibodies to the RSV F-protein havingsimilar properties may be obtained by one skilled in the art absentundue experimentation. These antibodies are distinct given the fact thatmost were generated in two different experiments, using different SCIDmice. The cell lines which express RF-1 and RF-2 have been maintained inculture for prolonged time, i.e., about 18 and 16 months respectively;dividing with an approximate doubling time of about 36-48 hours. Thespecific antibody concentration is on average about 0.8-1 μg/ml in aculture seeded at 0.5×10⁶ cells/ml grown for three days.

As discussed in greater detail infra, both RF-1 and RF-2 are IgG (1, k)with half-maximal binding to F-protein in ELISA at 0.6 and 1 ng/mlrespectively, and exhibiting isoelectric points of about 8.8 and 8.9respectively.

Moreover, these antibodies exhibit high affinity to the RSV F-protein.Specifically, for RF-1 the dissociation constant for RF-1 as determinedby plasmin resonance on an IASYS machine is about 10⁻¹⁰ M. The Kaconstant for RF-2 is similarly high; when determined by titrationmicrocolorimetry according to Wiseman et al. (1989) and Robert et al.(1989) it is about 2×10⁻⁹ M.

Additionally, these antibodies have been demonstrated to effectivelybind RSV infected cells, while not binding normal human cells tested,e.g., respiratory tract lining (HEp-2, a laryngeal epidermoid carcinoma,CCL 23), liver (HepG2, a human hepatoma cell line, HB 8068), lymphoidtissue, SB, a human B lymphoblastoid cell line, cat. no. CCL 120 andHSB, a T lymphoblastoid line, cat. no. CCL 120.1, and prostrate(LNCaP.FGC, a human prostrate adenocarcinoma line, cat. no. CRL 1740).

Significantly, both RF-1 and RF-2 both have been shown to exhibitsubstantial in vitro RSV viral neutralization. This was demonstrated intwo different assays (described in greater detail infra), i.e., aninfection neutralization assay effected by pre-reacting the virus withpurified monoclonal antibody prior to its addition to cells (whichmeasures ability of antibody to inhibit virus infectivity) and a fusioninhibition assay which measures the ability of the monoclonal antibodyto inhibit virus growth and expansion after virus entry into the cell.

Moreover, as discussed in greater detail infra, both RF-1 and RF-2inhibited virus infection of twelve different isolates at concentrationsrespectively ranging from about 30 ng/ml to 1000 ng/ml. Thus, RF-2apparently performs better than RF-1, yielding to 50% virus inhibition(ED50) at concentrations which are about 1.25 to 10 times lower thanRF-1.

By contrast, higher concentrations of monoclonal antibody are requiredto inhibit fusion and viral antigen expression in previously infectedcells, with RF-1 being about 5 to 10 times more potent than RF-2.Moreover, both RF-1 and RF-2 were effective against a Type B RSV, Type Bprototype RS6556, and a Type A RSV, Type A prototype R S Long. Thus, thein vitro results indicate that the subject human monoclonal antibodiesmay be used to treat or prevent RSV infection caused by different RSVstrains, both of Type A and Type B prototype. As discussed previously,the RSV F-protein is fairly well conserved in different RSV isolates.Therefore, it is likely that the subject monoclonal antibodies to theRSV F-protein bind to a conserved epitope of RSV F-protein.

As discussed, the subject human monoclonal antibodies or recombinanthuman antibodies containing the variable heavy and light sequencestherefrom (preparation discussed infra) will be used as therapeutic andprophylactic agents to treat or prevent RSV infection by passiveantibody therapy. In particular, the DNA sequence encoding these DNAvariable domains may be incorporated in IDEC's proprietary expressionvector which is depicted in FIG. 2. This version is substantiallydescribed in commonly assigned U.S. Ser. No. 08/379,072, (now pending)filed on Jan. 25, 1995, herein incorporated by reference. This vectorconstant human constant domain, for example, human gamma 1, human gamma4 or a mutated form thereof referred to as gamma 4 PE. (See U.S. Ser.No. 08/379,072, incorporated by reference herein.) In general, this willcomprise administering a therapeutically or prophylactically effectiveamount of the subject human monoclonal antibodies to a susceptiblesubject or one exhibiting RSV infection. A dosage effective amount willpreferably range from about 50 to 20,000 μg/Kg, more preferably fromabout 100 to 5000 μg/Kg. However, suitable dosages will vary dependenton factors such as the condition of the treated host, weight, etc.Suitable effective dosages may be determined by those skilled in theart.

The subject human monoclonal antibodies may be administered by any modeof administration suitable for administering antibodies. Typically, thesubject antibodies will be administered by injection, e.g., intravenous,intramuscular, or intraperitoneal injection, or more preferably byaerosol. As previously noted, aerosol administration is particularlypreferred if the subjects treated comprise newborn infants.

Formulation of antibodies in pharmaceutically acceptable form may beeffected by known methods, using known pharmaceutical carriers andexcipients. Suitable carriers and excipients include by way of examplebuffered saline, bovine serum albumin, etc.

Moreover, the subject antibodies, given their high specificity andaffinity to RSV infected cells possess utility as immunoprobes fordiagnosis of RSV infection. This will generally comprise taking asample, e.g. respiratory fluid, of a person suspected of having RSVinfection and incubating the sample with the subject human monoclonalantibodies to detect the presence of RSV infected cells.

This will involve directly or indirectly labeling the subject humanantibodies with a reporter molecule which provides for detection ofhuman monoclonal antibody-RSV immune complexes. Examples of known labelsinclude by way of example enzymes, e.g. β-lactamase, luciferase, etc.and radiolabels.

Methods for effecting immunodetection of antigens using monoclonalantibodies are well known in the art. Also, the subject anti-RSVF-protein antibodies in combination with a diagnostically effectiveamount of a suitable reporter molecule may be formulated as a test kitfor detection of RSV infection.

MATERIALS AND METHODS

The following Materials and Methods were used in Examples 1 to 6.

F protein preparation and purification:

F protein was prepared essentially according to the method of Walsh etal. J. Gen. Virol, 70, 2953-2961, (1989). Briefly, HEp-2 cells at 70%confluency were infected with the Long strain of RSV, a lab adaptedstrain of the A type. After culture for 48 hours in T-150 culture flasksin IMDM supplemented with 5% fetal calf serum, 2 mM glutamine and 2 mMsodium pyruvate, the cells were lysed in a lysing buffer of PBScontaining 1% Triton X-100 and 1% deoxycholate. F protein was purifiedfrom the crude cell lysate on an affinity column of Sephadex coupled toa murine monoclonal anti-F antibody, B4 (a kind gift from HiroykiTsutsumi) (Tsutsuni et al. 1987). The column was washed extensively withlysing buffer and purified F protein was eluted in 0.1 M glycine pH 2.5,containing 0.1% deoxycholate. The eluate was neutralized immediatelywith 1 M Tris, pH 8.5 and dialyzed against PBS. After the detergent wasremoved on a Extracti-D gel column (Pierce, Rockford, Ill., Cat. No.20346), F protein concentration was determined by EIA and the solutionwas sterilized by gamma irradiation.

Lymphoid cell preparation:

Spleen was obtained following clinically indicated splenectomy of anidiopathic trombopenic purpura (ITP) patient. A single cell suspensionwas prepared by sieving through a metal mesh, and washed in IMDM mediasupplemented with 2% fetal calf serum. Red blood cells were eliminatedby treatment with ammonium chloride lysing buffer for 90 seconds at 37°C. The white blood cell enriched suspension was then washed twice withserum containing media, resuspended in ice cold freezing media (95% FCSwith 5% DMSO) at 10⁸ cells/ml and frozen in liquid nitrogen until use.

In vitro immunization (IVI):

Cultures were set-up in IMDM supplemented with 2 mM glutamine, 2 mMsodium pyruvate, non-essential amino acids, 25 μg/ml IL-2 and 10% fetalcalf serum. An antibiotics cocktail was added including 2.5 μg/mlamphotericin, 100 μg/ml ampicillin, 100 μg/ml kanamycin, 5 μg/mlchlortetracycline, 50 μg/ml neomycin and 50 μg/ml gentamicin. The cellswere cultured in 6-well clusters at 3×10⁶ cells/ml with 40 ng/ml Fprotein. After three days, the cells were collected, washed andresuspended in HBSS at 8×10⁸ cells/ml for SCID reconstitution.

Reconstitution of SCID mice:

Five to eight week old female CB17/SCID mice were reconstituted byintraperitoneal injection of 200 μl of HBSS containing 4×10⁷ humanspleen cells subjected to M; the mice were boosted one week later ipwith 5 μg F protein in CFA and tail bled after another 15 days. Theirserum was tested for human IgG convention and anti-F protein antibodytiter.

Recovery of human cells from hu/SCID mice:

Two hu-SPL-SCID mice with high anti-F human antibody titers developedlarge abdominal tumors. Tumors were recovered by excision fromsacrificed mice under sterile conditions, single cell suspensions wereprepared, the cells were washed with IMDM containing 2% fetal calf serumand cultured at 10⁶ /cells ml in T-25 flasks in IMDM with 10% FCS.

Testing for human IgG and anti-F protein antibodies:

The testing for human IgG and anti-F antibodies was performed in ELISA.For that purpose, plates were coated overnight with GAH-Ig (0.05μg/well) or F protein (0.05 μg/well) respectively in 0.1 M bicarbonatebuffer, pH 9.5 and blocked with PBS containing 1% fetal calf serum.Serial dilutions of mouse sera, culture supernatants or purifiedantibodies were reacted to the plate. Bound human IgG were revealed bythe subsequent addition of GAH IgG-HRP and OPD substrate (Sigma.).Selected high titer human serum was used as a positive control in bothassays and purified polyclonal human IgG, or (γ, κ) myeloma protein wereused as a standard in the estimation of the concentration of human IgGand monoclonal antibodies respectively.

Isotyping of human antibodies:

Isotyping was performed in ELISA on F protein coated plates as describedabove. Bound human IgG were revealed by the subsequent addition of HRPconjugated mouse monoclonal antibodies specific for human γ1, γ2, γ3,γ4, μ, κ and λ chains. Positive controls were run with myeloma proteinsof the (γ1, κ), (γ2, κ), (γ3, λ), (γ4, λ) or (λ, λ) isotype and free κand λ chains.

Protein A purification:

Antibodies were purified from culture supernatants on a proteinA-Sepharose 4B column. Briefly, supernatants were collected, filteredthrough 0.2 mm filters and supplemented with 0.02% sodium azide. Columns(gel volume approximately 0.5 ml) were equilibrated in PBS with 0.02%sodium azide, then loaded with supernatant at low speed. After extensivewashing, bound human monoclonal IgG were eluted in 0.1 M sodium citratebuffer, pH 3.5, dialyzed against PBS-azide using Centricon 10 filters(Amicon) and sterilized by gamma irradiation until further use. Columnswere regenerated with citric acid pH 2.5 and re-equilibrated with PBSwith 0.02% sodium azide for subsequent use.

Isoelectric focusing:

Isoelectric focusing (IEF) of human antibodies was performed inpolyacrylamide pre casted gels (Pharmacia, Uppsala, Sweden, Cat. No.80-1124-80), pH 3-pH 10. Briefly, 20 μl of samples were loaded and runat 1500 volts for 90 minutes. Standards of pi 5.8 to 10.25 were used forpi reference. Gels were stained in Coomassie blue stain and destained indestaining buffer containing 25% methanol, 68% water and 8% acetic acid.

Western Blot:

Purified F protein, both native and denatured by boiling, was migratedin a 10% polyacrylamide gel. The gel was blotted on a nitrocellulosesheet at 30 volts for 2 hours and 60 volts overnight. After transfer,the nitrocellulose was blocked for 1 hour at room temperature with 1%BSA and 0.1% Tween-20 in PBS. Different strips were washed in PBS andthe primary antibodies, hu-SPL-SCID anti-F protein sera, or hu-SPL-SCMDanti-tetanus toxoid negative control, or mouse anti-F protein positivecontrol, were added for 1 hour. All sera were diluted 1:500. Afterextensive wash with PBS, the secondary antibody, GAH IgG-HRP for thesamples and the negative control, or GAM IgG for the positive control,was added for 1 hour. Blots were revealed with 4-chloro-1-naphtol.

Immunofluorescence:

RSV infected HEp-2 cells (4×10⁴) were fixed on glass slides using icecold acetone and were reacted with 20 μl of serum diluted 1:10 orpurified MAb, 2 μg/ml, for 1 hour at 37° C. The slides were washed andthe bound antibodies were revealed with GAH IgG-FITC, for 30 minutes at37° C. and observed under a fluorescence microscope.

FACScan analysis:

RSV-infected HEp-2 cells (10⁶ cells/sample) were washed with washingbuffer (PBS with sodium azide 0.1%). The cell pellet was suspended in 50μl of incubation buffer (PBS with sodium azide 0.1% supplemented withBSA 0.1%) containing 2 μg/ml RF-1 or RF-2. After 15 minutes incubationon ice, the cells were washed and resuspended in incubation buffercontaining GAH IgG-FITC for another 15 minutes on ice. After 3 washes,the cells were fixed in 1 ml PBS with 1% formaldehyde and analyzed in aBecton-Dickinson FACScan apparatus.

Affinity determination:

Two methods were used to determine the affinity of human MAbs to solubleF protein:

In plasmon resonance, using an IASYS machine, antibody was boundcovalenty to the wet side of a device from which the change in mass canbe determined based on the change of refraction of light shone on thedry side of the device. Different concentrations of F-protein were addedand subsequently eluted off with a steady flow of PBS. The change inmass as a result of F-protein release from the antibody was measured,and from the kinetics a K_(off) was determined. Ka was calculated bytesting the off-rate from different levels of initial saturation.

Alternatively, affinity constant was determined by micro-calorimetryaccording to Wiseman et al and Robert et al., as follows: RF-2 and Fprotein were co-incubated at a known concentration in a thermo-chamberat 42° C. and the enthalpy change due to the immune complex formation inthe solution was measured. The reaction was repeated at 50° C. Thebinding association constant K was calculated as a function oftemperature and enthalpy change according to Robert et al. in thefollowing equation:

    K=Kobs.e.sup.ΔHobs/R.(1/T-1/Tobs). e.sup.ΔCTobs/R.(1/T-1/TTobs). (T/Tobs).sup.ΔC/R

where Kobs is the binding equilibrium constant and ΔH^(obs) is theenthalpy change observed experimentally, at a given absolutetemperature, Tobs; R is the universal gas constant (1.987) and ΔC is theexperimentally determined binding heat capacity change.

Complement-enhanced virus neutralization assay:

Two laboratory strains (Long, type A and 18537, type B) and ten wildtype RS virus isolates, which were isolated from hospitalized infants,were used to assess the neutralizing capacity of ant-F protein humanMAbs. Serial dilutions of human MAb were pre incubated with virus(50-100 pfu) in the presence of complement for 30 minutes at roomtemperature, in 100 μl IMDM/well of microtitration plate. HEp-2 cells(5×10⁴ /well) were added in 100 μl MEM and incubated for 3 days at 37°C., 5% CO₂. The plates were washed, fixed with acetone and air dried andRSV antigen was detected by ELISA using mouse MAbs. The neutralizationend point was determined arbitrarily as the dilution which reducedantigen production by 50% compared to control wells with no antibody.

Virus fusion inhibition assay:

Fusion inhibition titers were determined by pre incubating 100 TCID₅₀RSV S Long (prototype A virus) or RS 6556 (Type B clinical isolate) withVERO cells (5×10³ /well) in microtitration plates, for 4 hours at 37°C., 5% CO2. Various concentrations of human monoclonal antibodies orcontrols were added to each well and quadruplicate cultures wereincubated for 6 days at 37° C., 5% CO₂. Control cultures contained virusnon infected cells (negative) or infected cells in the absence ofantibody (positive). Virus growth was detected in ELISA using rabbitpolyclonal anti-F protein antisera and HRP-labelled anti-rabbit IgG. Thereaction was developed with IMBlue substrate (KPI, Gaithersburg, Md.).Titers (ED50) were defined as the concentration of antibody inhibitingvirus growth by 50% based on regression analysis of the MAb doseresponse.

EXAMPLE 1

HU-SPL-SCID titers:

Fifteen SCmD mice received human spleen cells from a single donor with mcondition. The cells were previously cultured for three days in thepresence of IL-2 and different concentrations of soluble F protein. Allanimals were successfully reconstituted and, after boost with F protein,total human IgG concentrations varied from 12 μg/ml to 10 mg/ml in theserum and anti-F protein titers varied from 3×10² to 10⁶ (Table I). Nocorrelation was observed between in vitro F protein exposure and anti-Fprotein titer in vivo. It has been previously observed with the subjectmethod, in the horse ferritin antigen system, that antigen exposure isnecessary during in vitro cultivation of the spleen cells tosubsequently ensure specific antibody titer in vivo. The discrepancybetween these two systems may be attributed to the difference in theantigens involved: since humans are not-naturally exposed to horseferritin, the IVI step involves an antigen priming of the spleen cellsand induces a primary response in vitro; on the other hand, virtuallyall humans are immune to RSV through natural infection in early life,which leads to a permanent memory to F protein, therefore stimulationwith IL-2 alone in vitro followed by one boost in vivo is enough toinduce secondary responses.

The antisera were polyclonal, as judged from isoelectric focusingpatterns (data not shown). They were tested for reactivity to F proteinin Western blot. Our results showed that polyclonal human Abs didrecognize soluble native F protein both in its dimer form (140 KD) andits monomer form (70 KD); they also reacted strongly with denatured Fprotein, binding specifically to the 2 subunits of 48 KD and 23 KD(representative data in FIG. 1). This suggests that at least a fractionof the humoral response to F protein is directed against linear, nonconformational epitopes of the molecule. Immunofluorescence studiesfurther demonstrated the specificity of the hu-SPL-SCID sera, sinceimmune sera, but not naive SCID mouse sera, reacted strongly withRSV-infected HEp-2 cells (FIG. 2). No reactivity was observed towardsnoninfected HEp-2 cells used as negative control. It was concludedtherefore that soluble F protein was an adequate antigen for thegeneration of antibodies specific to the membrane viral antigenexpressed on naturally infected cells.

EXAMPLE 2

Identification of antibodies in tumor cell cultures:

All mice with high anti-F protein titers were sacrificed and human cellswere harvested from peritoneal lavage and spleens. Two mice (hu-SPL-SCID#6 and hu-SPL-SCID #15) spontaneously developed abdominal solid tumorsthat were recovered and teased into single cell suspension. The tumorcells secreted specific anti-F protein antibodies as determined inELISA. These tumors and antibodies are referred to as RF-1 (ESVE-protein) and RF-2. RF-1 and RF-2 were generated in two differentexperiments separated by approximately two months and were isolated fromindividual hu-SPL-SCID mice, and are thus distinct antibodies; they haveestablished themselves in culture for more than 18 months and 16 monthsrespectively, dividing with an approximate doubling time of 36-48 hours.Specific antibody concentration is typically of 0.5-1 μg/ml in a cultureseeded at 0.5×10⁶ cells/ml and grown for three days.

For further characterization, both human MAbs were purified from culturesupernatants by affinity chromatography, using Protein A Sepharosecolumns. Both RF-1 and RF-2 are IgG(_(l),k), with half maximal bindingto F-protein in ELISA at 0.6 and 1 ng/ml respectively (FIG. 3). From themigration pattern in IEF, RF-1 and RF-2 isoelectric points weredetermined to be 8.8 and 8.9 respectively (FIG. 4). RF-1 and RF-2specifically recognized RSV infected HEp-2 cells in flow cytometry (FIG.5). The dissociation constant, Kd, for RF-1 was determined by plasmonresonance on an LASYS machine to be in the 10⁻¹⁰ M range. The Kdconstant of RF-2 was determined by titration micro calorimetry,according to Wiseman et al (1989) and Robert et al. (1989) to be 2×10⁻⁹M.

EXAMPLE 3

Tissue specificity of anti-F-protein:

Purified antibodies were screened for reactivity to a series of humancell lines available at ATCC by means of indirect immunofluorescenceassays measured by flow cytometry (Table II): The results showed thatthe antibodies did not bind to cell lines representing respiratory tractlining (HEp-2, a laryngeal epidermoid carcinoma, Cat. No. CCL 23), liver(HepG2, a human hepatoma cell line, Cat. No. HB 8065), lymphoid tissue(SB, a human B lymphoblastoid cell line, Cat. No. CCL 120 and HSB, a Tlymphoblastoid line, cat.no. CCL 120.1) and prostate (LNCAP.FGC, a humanprostate adenocarcinoma line, Cat. No. CRL 1740).

EXAMPLE 4

In vitro functional activity:

To determine whether the antibodies had virus neutralizing effect invitro, they were subjected to two types of functional assays: Infectionneutralization assays were performed by pre-reacting the virus withpurified MAb prior to its addition to the cells and therefore reflectthe ability of the MAb to inhibit virus infectivity; fusion inhibitionreflects the ability of the Ab to inhibit virus growth and expansionafter virus entry in the cell. The outcome of both assays was measuredas the amount of virus released in the culture after a given incubationdime, as determined by viral antigen titration in EIA.

Both Abs were able to inhibit virus infection, of all twelve isolatestested, at concentrations ranging from 30 ng/ml to 1000 ng/ml and from 8ng/ml to 165 ng/ml, for RF-1 and RF-2 respectively. RF-2 performedconsistently better than RF-1, yielding to 50% virus inhibition (ED50)at concentrations 1.25 to 10 times lower than RF-1. Representative dataare indicated in Table III.

As expected, higher concentrations of MAb were required to inhibitfusion and viral antigen expression in previously infected cells. Inthis assay, RF-1 was 5 to 10 times more potent than RF-2. Both MAb weremore effective in the Type B prototype RS 6556 than in the Type Aprototype R S Long (Table III).

                  TABLE I    ______________________________________                                 hu IgG    mouse #  Ag! in vitro                      fresh cells                                 (μg/ml)                                       anti-F titer    ______________________________________    1       1 μg/ml                      +          1,000 10.sup.6    2       1 μg/ml                      +          12.3  10.sup.3    3       1 μg/ml                      +          3,000 10.sup.6    4       1 μg/ml                      +          8,750 10.sup.6    5       1 μg/ml                      +          1,000 10.sup.6    6       1 μg/ml                      -          1,500 10.sup.5    7       1 μg/ml                      -          162   10.sup.5    8       1 μg/ml                      -          4,500 10.sup.6    9       1 μg/ml                      -          333   10.sup.5    10      40 ng/ml  -          3,300 5 × 10.sup.5    11      40 ng/ml  -          554   3 × 10.sup.2    12      1 μg/ml                      -          10,000                                       5 × 10.sup.5    13      1 μg/ml                      -          200   5 × 10.sup.4    14      0 μg/ml                      -          182   5 × 10.sup.4    15      0 μg/ml                      -          3,300 10.sup.5    ______________________________________     Table I: Splenocytes from a single donor were cultured in the presence of     IL2 for 3 days, with or without F protein. SCID mice were reconstituted     with 4 × 10.sup.7 cells and boosted with 10 μg of F protein ip i     CFA. In mice # 1, 2, 3, 4 and 5, fresh autologous cells (20 ×     10.sup.6) were injected with the boost. Human IgG concentration was     determined by comparison to a standard curve of polyclonal IgG and antiF     protein titer was determined by end point dilution in  # EIA.

                  TABLE II    ______________________________________    Cell line    Tissue Type     Tissue Labeling    ______________________________________    HEp-2        Laryngeal epidermis                                 -    RSV infected- HEp-2                 Laryngeal epidermis (RSV)                                 ++++    SB           Lymphoid        -    HSB          Lymphoid        -    LNCaP        Prostate        -    HepG2        Liver           -    ______________________________________     Table II: Reactivity of RF2 with various cell lines. Various cell lines     were subjected to indirect immunofluorescence labeling with RF2, 200     ng/10.sup.6 cells. A Fab goat antihuman IgGFITC was used as second step.     (-) indicates the presence of RF2 did not result in change of channel for     the average fluorescence; (+) indicated increase of average labeling by     0.5 log.

                  TABLE III    ______________________________________                               Infection Neutrali-    Fusion Inhibition activity zation Activity    ED.sub.50 titer            ED.sub.50 titer           RS Long   RS 6556         MR 144 18537    Antibody           (Type A)  (Type B) Antibody                                     (Type A)                                            (Type B)    ______________________________________    RF-1    660 ng/ml                      40 ng/ml                              RF-1   30 ng/ml                                            30 ng/ml    RF-2   3300 ng/ml                     400 ng/ml                              RF-2    8 ng/ml                                            12 ng/ml    ______________________________________     Table III: ED.sub.50 is defined as concentration of antibody inhibiting     virus growth by 50% based on regression analysis of the monoclonal     antibody doseresponse.

EXAMPLE 5 (COMPARATIVE)

Induction of IgG Recall Responses to F-protein in vitro:

More than 95% of the population over 2 years of age have been exposedto, and responded successfully to RSV Henderson et al, J. Med. (1979),300, 530-534. Challenge of spleen cell in vitro with RSV F-proteinshould, therefore, result in recall responses, and, indeed, mainly IgGresponses were induced in vitro with spleen cells (see FIG. 5). Theoptimal antigen concentration, 40 ng/ml, was at least one order ofmagnitude lower than what was observed for antigens inducing primaryresponses, i.e. ferritin, Ilig/ml, Boerner et al, J. Immunol., 1991,147, 86-95; Brams et al, Hum. Antibod. Hybrnomas, 1993, 4, 47-56.Therefore, it must be considered that in vitro priming with F-proteininduces secondary like responses. Several attempts to induce significantin vitro responses to RSV F-protein failed with PBMCs and tonsil derivedcells.

A limited effort to generate monoclonal antibodies from in vitro primedspleen cells resulted in several monoclonal IgG antibodies to RSVF-protein. Most of these, however, cross-reacted to one of severalcontrol antigens in ELISA (results not shown).

EXAMPLE 6

Cloning of the genes coding for RF-2:

Neither the RF-1 nor the RF-2 clone produce significant amounts ofantibody. Also, both of these cell lines grow best in media with 20%FCS, which is disadvantageous because it results in contamination of thepurified antibody with bovine IgG. Therefore, in order to be able toproduce and purify amounts of antibody necessary for doing meaningfulanimal model tests, which typically requires up to 1 gram of oneselected antibody, it is advantageous to transfer the genes coding forRF-1 and RF-2 to a production vector and cell line. The presentassignee, IDEC Pharmaceuticals, Inc., has developed a very efficienteukaryotic production system which results in the production of humanmonoclonal antibodies in CHO cells. This vector system is described incommonly assigned U.S. Ser. No. 08/379,072, (now pending) filed Jan. 25,1995, and in commonly assigned U.S. Ser. No. 08/149,099, filed Nov. 3,1993, both of which are incorporated by reference herein. Routinelyusing this system antibody gene transfected CHO cells produce around 200mg antibody per liter of serum free medium in spinner cultures andgreater than 500 mg/liter in fermentors after amplification inmethotrexate.

Cell culture cloned (see below) RF-2 cells, approximately 5×10⁶, weresubjected to RNA extraction using a mRNA isolation kit, Fat Track(InVitroGen, San Diego, Calif.), and single stranded cDNA was preparedusing an oligo-dT primer and reverse transcriptase. An aliquot of cDNAwas used as the starting material for polymerase chain reaction (PCR)amplification of the variable region genes. PCR was performed using twosets of primers SEQ ID NOS:2-5!. (see Table IV).

                                      TABLE IV*    __________________________________________________________________________    Heavy chain primers with Mlu 1 site    V.sub.H 1        5' (AG).sub.10 ACGCGTG(T/C)CCA(G/C)TCCCAGGT(G/C)CAGCTGGTG 3'    V.sub.H 2        5  (AG).sub.10 ACGCGTGTC(T/C)TGTCCCAGGT(A/G)CAG(C/T)TG(C/A)AG 3'    V.sub.H 3        5  (AG).sub.10 ACGCGTGTCCAGTGTGAGGTGCAGCTG 3'    V.sub.H 4        5  (AG).sub.10 ACGCGTGTCCTGTCCCAGGTGCAG 3'    V.sub.H 5        5  (AG).sub.10 ACGCGTGTCTGGCCGAAGTGCAGCTGGTG 3'    Heavy chain constant region primer  SEQ ID NO:25! anti-sense strand with    Nhe 1 site    IgGl-4        (AG).sub.10 GCCCTTGGTGCTAGCTGAGGAGACGG 3'    Kappa Chain primers  SEQ ID NO.: 26! with Dra III site    1.  5' (AG).sub.10 CCAGGTGCACGATGTGACATCCAGATGACC 3'    2.  5' (AG).sub.10 CCTGGATCACGATGTGATATTGTGATGAC 3'    3.  5' (AG).sub.10 CCAGATACACGATGTGAAATTGTGTTGAC 3'    4.  5' (AG).sub.10 TCTGGTGCACGATGTGACATCGTGATGAC 3'    Kappa constant region primer  SEQ ID NO.:27! anti-sense strand with Bsi    WI site    C.sub.k        5  (AG).sub.10 TGCAGCCACCGTACGTTTGATTTCCA(G/C)CTT 3'    __________________________________________________________________________     *Legend for Table IV: Synthetic oligonucleotide primers used for the PCR     amplification of human immunoglobulin heavy and light chain variable     regions. Restriction sites for cloning are underlined in bold.

The first set of primers was designed for amplifying the heavy chainvariable regions. It consists of one 3 primer that binds in the J regionand five family-specific 5' primers that bind in the late leader andframework 1 region. A second set of primers was designed for amplifyingthe Kapp variable region. It consists of one 3' primer and four 5'primers that bind in the late leader and framework 1 regions. The PCRreactions were electrophoresed on agarose gels and correctly sized 350base pair bands were excised. The DNA was electroeluted, cut withappropriate restriction enzymes and cloned into IDEC's NEOSPLAexpression vector. (See FIG. 6) The NEOSPLA vector used for expressionof human antibodies contains the following: CMV=cytomegaloviruspromoter, BETA mouse beta globin major promoter, BGH=bovine growthhormone polyadenylation signal, SVO=SV40 origin of replication.N1=Neomycin phosphoamsferase exon 1, N2=Neomycin phosphotansferase exon2. UGHT=Human ininuoglobulin kappa constant region. Heavy=Humanimmunoglobulin gamma 1 or gamma 4 PE constant region. L=leader. SV=SV40polyadenylation region.

IDEC's NEOSPLA expression vectors were designed for large scaleproduction of immoglobulin genes (See, Reff et al, Blood, (1994), 83,435-445, incorporated by reference in its entirety). Mouse/humanchimerics, primate/human chimerics and human antibodies have beensuccessfully expressed at high levels using these vectors. NEOSPLAcontains a neomycin phosphotransferase gene for selection of CHO cellsthat have stably integrated the plasmid vector DNA. In addition, NEOSPLAconnnns a dihydrofolate reductase gene for amplification inmethotrexate, a human constant light chain (either κ or λ) and a humanconstant heavy chain region (either γ1 or γ4(PE)). Gamma 4 (PE) is thehuman γ4 constant region with 2 mutations, a glutamic acid in the CH2region which was introduced to eliminate residual FcR binding, and aproline substitution in the hinge region, intended to enhance thestability of the heavy chain disulfide bond interaction, Algre et al, J.Immunol., 148, 3461-3468, (1992); Angal et al, Mol. Immunol, 30, 105-108(1993), both of which are incorporated by reference herein. Uniquerestriction sites have been incorporated into the vector in order tofacilitate insertion of the desired and light variable regions. Reff etal., Blood, (1994), 83, 435-445.

The light chain of RF-2 has been cloned into NEOSPLA in duplicate andsequenced following the method of Sanger et al. Sanger et al., Proc.Natl. Acad. Sci. (1977), 74, 5463-5467. The kappa chain is a member ofthe kappa 2 subgroup. Similarly, the human heavy chain variable regionof RF-2 has been isolated and cloned in front of the human γ1 consantdomain.

The light chain coding genes of RF-1 and RF-2 were readily cloned,whereas cDNA for the genes encoding the heavy chains could not begenerated using the common Tac reverse transcriptase. However, thisproblem was obviated by substituting a high temperature, 70° C., reversetranscriptase. Thereby, intact PCR products could be generated withprimers primarily derived from V_(H) 2 family genes.

The amino acid sequence and the nucleic acid sequence for the RF-1 lightand heavy variable domains may be found in FIGS. 7a and 7b,respectively. The amino acid sequence and the nucleic acid sequence forthe light and heavy variable domains for RF-2 may be found in FIGS. 8aand 8B, respectively. FIGS. 9a-9c depict the nucleic acid and amino acidsequence of RF-1 as expressed in the subject NEOSPLA vector. FIGS. 9aand 9b depict the leader, variable light and heavy, and human constantdomain sequences, i.e., the human kappa domain and the humangamma/constant domain. FIG. 9c shows the amino acid and nucleic acidsequence of the human gamma/constant domain. FIG. 10 depictsschematically an expression vector which provides for the expression ofthe sequences set forth in FIGS. 9a-9c and thereby recombinant RF-1 inCHO cells.

FIGS. 11a-11a similarly depict the amino acid and nucleic acid sequencesof the leader sequence, RF-1 variable light, human kappa constantregion, RF-2 variable heavy, and human gamma/constant domain. FIG. 12depicts schematically an expression vector which provides for theexpression of recombinant RF-2 in CHO cells.

EXAMPLE 7

Development of a protocol for cloning of EBV transformed cells:

Antibody production from EBV transformed cells continuously decrease,and ultimately ceases. Kozbor et al., J. Immunol. (1981), 127,1275-1280. To immortalize the antibody production, it is thereforeessential to extract the immunoglobulin coding genes from the cellsbefore this event and transfer those into an appropriate expressionsystem. In order to isolate the genes coding for the antigen bindingvariable domains of antibodies produced by EBV transformed cells, it isessential to ensure that the cell material is monoclonal. EBVtransformed cells are, however, very difficult to clone whether bylimiting dilution or in semisolid agar. Isoelectric focusing gelelectrophoresis of protein A purified preparations of our two anti-Fprotein antibodies, RF-1 and RF-2, showed at least two populations ofantibodies in the RF-2 preparation and the possibility of oligoclonalityin the RF-1 preparation.

By using the mouse thyoma line EL4 BS Zhang et al, J. Immuno., (1990),144, 2955-2960, as feeder layer, cells were expanded from a single cellthrough limiting dilution. The human thyoma cell line EL4 BS expressesgp39 in a membrane receptor way that induces B cells to grow. 5×10⁴ EL-4B5 cells/well were plated out in a microliter plate, and cells from thecultures were plated out on the EL-4 B5 layer at various concentrations,from 0.38 cells/well and up. The number of wells with growth for each ofthe concentration plated were counted after an appropriate amount oftime.

The supernatant was tested for presence of hnman IgG and forantigen-specific IgG. With this protocol we have isolated and cloned thecells that produce RF-1 (see Table V) and RF-2 (see Table VI),respectively, from the original oligoclonal preparations. Thenon-specific antibodies found in the cloning were only analyzed withrespect to isotype, and were found to be the same as the specificantibodies, IgG1k. Based on the yield of F-protein specific clones fromfreezes made at various time points during the cultivation of RF-1, aswell as the amount of IgG that was produced, it was estimated that thespecific antibody made up approximately 1/20 of the total antibodyamount shortly after the start of the culture and disappeared afterapproximately 8 months in culture. RF-2 made up a much higher part ofthe total IgG, no less than 10% at any given time. Antibody from theoligoclonal preparations was used to generate the in vitroneutralization data, resulting in an overestimation of the ED50 titers.Our affinity studies with plasmon resonance, however, were not dependenton using pure antibodies. The affmity studies using titration microcalorimetry-was done with cloned material.

                  TABLE V*    ______________________________________    # cells/well             # wells # anti-F wells (%)                                  # wells with growth (%)    ______________________________________    30       48       48(100)      48(100)    10       48       48(100)      48(100)    3.3      96       27(28)       68(71)    1.1      192     17(9)        112(58)    0.38     384     18(5)        116(30)    ______________________________________     *Legend for Table V: Cloning of RF1 by limiting dilution. EL4B5 cells wer     plated out at 5 × 10.sup.4 cells/well in a flat bottomed 96 well     plate. Approximately 24 hours later, RF1 cells in exponential growth were     plated out on the feeder layer at the described concentrations. After 2-3     weeks, the wells were scored for growth and for presence of antiF     activity.

                  TABLE VI*    ______________________________________    # cells/well             # wells # anti-F wells (%)                                  # wells with growth (%)    ______________________________________    30        40     40(100)      15(37.5)    10       120     120(100)     22(18)    3.3      120     102(85)      9(7.5)    1.1      120     50(41.6)      1(0.83)    .33      180     30(16.7)     5(2.8)    ______________________________________     *Legend for Table XIV: Cloning of RF2 by limiting dilutio/n. Done as in     Table VIII.

In order to confirm the clinical applicability of the two humanmonoclonal antibodies with in vitro virus neutralizing activity, theseantibodies are flier characterized with respect to their efficacy inclearing RSV infection in two different animal models. These preclinicalperformance evaluations are effected with material produced by CHO cellstransfected with the cloned genes coding for the antibodies insertedinto a proprietary expression vector (see FIG. 6). Two antibody models,one with intact complement and Fc receptor binding domains, γ1, and onevoid of these domains, γ4 (PE mutant), Alegre et al., J. Imnunol.,(1992), 148, 3461-3468; Angal et al., Molecular Immunonolog, (1993), 30,105-108, will be tested. The rationale for testing γ4 version is basedon two considerations: (i) Anti-F-protein Fabs have shown significantvirus neutralizing effect in vitro Barbas et al., Proc. Natl. Acad. Sci.(1992), 89, 10164-10168, as well as in vivo, Crowe et al, Proc. Natl.Acad. Sci. (1994), 91, 1386-1390, albeit when administered directly intothe lung, (ii) potentially avoiding lung damage caused by effectorfinction activation in sensitive tissue already stressed by virusinfection could be advantageous. A set of nonspecific controlantibodies, one γ1 and one γ4 (PE), will be generated from an in-housenon-specific hybridoma IgG₁ antibody.

The first animal model is a mouse model, Taylor et al., J. Immunology(1984), 52, 137-142; Walsh, E. E., J. Infectious Diseases (1994), 170,345-350. This model is used to determine the effective dose, defined asthe smallest dose resulting in a 2 log reduction in virus load in thelung tissue after 1 weeks incubation. This model is also used todetermine which of the antibody models to proceed with. The secondanimal model is a primate model using the African green monkey, Kakuk etal, J. Infectious Diseases (1993), 167, 553-561. RSV causes lung damagein the African green monkey,. and this model's main purpose is forevaluating the damage preventing properties of the antibodies. Thenumber of tests with this model will be limited to test one antibody in5 different doses. The antibody, the dose and the infusion date relativeto infection date will be determined based on the findings with themouse model. Lung section is examined for virus load in plaque assay andby light microscopy for detection of lesions caused by RSV.

It will be observed if any changes in the amino acid sequence have takenplace during the process of stimulating and expanding the cells thatproduce the two antibodies. This is done by testing with a set of PCRprimers based on the CDR3 regions of theavy chains of RF-1 and RF-2whether the sequences of the genes coding for RF-I and RF-2 are presentin the original frozen cell material from which the two cell lines weregenerated. The positive control is RF-1 and RF-2 spiked source cells.The analysis follows the principles established by Levy et al., 1989,Levy et al., J. Exp. Med. (1989), 169:2007 and Alegre et al., J.Immunol. (1992), 148, 3461-3468; Angal et al., Molecular Immunology(1993), 30, 105-108; Foote et al., Nature (1991), 352, 530-532; Rada etal., Proc. Natl. Acad. Sci. (1991), 88, 5508-5512; Kocks et al., Rev.Immunol. (1989), 7, 537-559; Wysocki et al., Proc. Natl. Acad. Sci.(1986), 83, 1847-1851; Kipps et al., J. Exp. Med. (1990), 171, 189-196;Ueki et al., Exp. Med. (1990), 171, 19-34.

EXAMPLE 8

Generate CHO cell lines that produce large amounts (>100 mg/liter) ofRF-1 and RF2:

a. Transfect expression plasmids, isolate and expand G418 resistant CHOclones expressing the highest levels of RF-1 and RF-2.

Once the RF-1 and RIF-2 variable region genes are cloned into NEOSPLA,Chinese hamster ovary (CHO) cells (DG44), Urlaub et al, J. Somat. CellMol Genet., (1986), 16, 555, are transformed with the plasmid DNA. CHOcells are grown in SSFM H minus hypoxanthine and thymidine (G1BCO).4×10⁶ cells are electroporated with 25 μg plasmid DNA using a BTX 600electroporation device (BTX, San Diego, Calif.) in 0.4 ml disposablecuvettes. Prior to electroporation, the plasmid DNA will be restrictedwith Pac I which separates the genes expressed in mammalian cells fromthe portion of the plasmid used for growth in bacteria. Conditions forelectroporation are 230 volts, 400 micro faradays, 13 ohms. Eachelectroporation is plated into a 96 well dish (about 40,000 cells/well).Dishes are fed with media containing G418 (Geneticin, GIBCO) at 400μg/ml three days following electroporation, and thereafter,periodically, until colonies arise. Supernatant from colonies is assayedin ELISA for the presence of human IgG and for anti-F-protein activity.

b. Amplify the expression of antibody in methotrexate.

The G418 resistant colonies producing the highest amount ofimmunoglobulin are then transferred to larger vessels and expanded. Theexpression of the highest secreting G418 clone is increased by geneamplification, by selection in 5 nM methotrexate (MTX) in 96 welldishes. The 5 nM colonies producing the highest amount of antibody arethen expanded and then expression amplified again by selection in 50 nMMTX in 96 well dishes. Following this protocol, we have previously beenable to derive CHO cells that secrete greater then 200 mgs/liter in 7days in spinner culture (greater than 0.5 gram/liter in fermentors in 6days). Human antibody is then purified from supernatant using protein Aaffinity chromatography.

c. Produce and purify antibody.

100 mg of each antibody is generated. The selected antibody is producedin amounts determined from the mouse model studies. Spinner flasks withselected CHO transfectomas in CHO-S SFM II serum free medium (GIBCO Cat.No. 91-0456DK) with 50 nM methotrexate are used to produce antibody inthe required amounts. Supernatant are harvested and filtered through aset of filters to remove particular material, ending up with a 0.2 nmfilter. The supernatant is run through a protein A column with apredetermined size based on the total amount of antibody. After washing,the antibody is eluted from the column with 0.1 M Glycine/HCI, pH 2.8,into a neutralization buffer, 1 M Tris./HCI, pH 7.4. Theelution/neutralization buffer is exchanged extensively, ≧1000 times,with sterile PBS by ultrafiltration through an Amicon Centriprep orCentricon 30 (Cat. no. 4306 and 4209). The concentration of antibody isadjusted to 2 mglml and sterilized by filtration through a 0.2 nmfilter. The antibody is purified and stored on ice in 2 ml cryotubesuntil use in animals.

EXAMPLE 9

Characterize RF-1 and RF-2 in respect to performance in RSV animalmodels:

The performance of RF-1 and RF-2 is determined using appropriate animalmodels. The evaluation is divided into two steps, first (a) a Balb/cmodel, Taylor et al., J. Immunology (1984), 52, 137-142; Crowe et al.,Proc. Natl. Acad. Sci. (1994), 91, 1396-1390; Connors et al., J.Virology (1992), 66, 7444-7451, to determine the potency of theantibodies, as well as to determine what type of support (effector)functions are essential for the antibody to clear the virus load. Fromthe data gained in the mouse model, one candidate is chosen for furtherstudies in a primate model. The primate model is an African green monkeymodel, Kakuk et al., J. Infectious Diseases (1993), 167, 553-561. Aprimate model is especially suitable for confirming that the subjectmonoclonal antibodies can be used to prevent virus associated lungdamage.

a. Test performance in mouse model.

The rodent-model we have chosen is the Balb/c mouse. This model is wellcharacterized, Crowe et al., Proc. Natl. Acad. Sci. (1994), 91,1386-1390; Connors et al., J. Virology (1992), 66, 7444-7451, forstudies on passive therapy studies. Balb/c mice are highly permissive togrowth of RSV in both upper and lower airways at all ages, Taylor etal., J. Immunolog (1984), 52, 137-142. Animals are housed in groups of5, fed standard mouse chow and water ad lithium and cared for accordingto the Rochester General Hospital vivarium guidelines. These guidelinesare in compliance with the New York State Health Department, the FederalAnimal Welfare Act and DHHS regulations. All procedures, includinginjections, virus infection, orbital bleeding and sacrifice by cervicaldislocation, are performed under penthrane anesthesia in a vented hood.

i. Determine effective dose of antibody and compare performance of γ1and γ4 (PE version) of RF-I and RF-2.

Groups of 5 mice are infected by intranasal instillation of 10⁶ Long(subgroup A) or 10⁵ 18537 (subgroup B) plaque forming units (PFU) of RSVin a 100 μL volume on day 0. On day four, at peak virus titer, animalswill be injected intraperitoneally with each of the four F-proteinspecific monoclonal antibody preparations or control antibody. The dosestested are initially centered around a reference dose calculated toprovide a serum neutralization titer of approximately 1:300 or greaterfrom in vitro studies. This titer has been associated with protectivelevels against challenge with RSV in small animals. Dose response isevaluated by treatment with 25, 5, 1, 1/5 and 1/25 of the referencedose. Experiments with higher or lower doses are performed if warranted.Control mice are injected with an equivalent dose of the isotype matchedmonoclonal antibodies, as described above. Twenty-four hours later; day5 is the peak of virus shedding, the mice are sacrificed. Serum isobtained by intracardiac puncture and the nasal turbinates and lungs areremoved, weighed and homogenized in 1 and 2 ml of NMM, respectively.Homogenates are titered for virus on HEp-2 cells, and virus titersexpressed as TCID₅₀ /gm tissue. The mean titers between groups arecompared to the control group by the student t-test. Serum is obtainedat the time of infection and at sacrifice for human monoclonal anybodyquantification by enzyme immunoassay and neutralization assay. It isanticipated that the greatest reductions in virus titer will be in lungvirus growth since IgG isotypes are not actively secreted (in contrastto IgA) in the upper respiratory tree. Should therapy on day 4 ofinfection prove ineffective at reducing lung virus, therapy on days 2and/or 3 will be assessed.

The titer of each monoclonal antibody in stock solutions and in serumfrom injected animals is determined using an ELISA,as described supra.RSV fusion protein purified by affinity chromatography according toestablished methods Walsh et al., J. Gen. Virol. (1985), 66:409-415 isused in the solid phase. A separate assay for the RSV G protein willalso be devised for evaluation of mouse IgG responses to experimentalRSV infection. Rabbit antibody specific for human IgG and mouse IgG(available from Virion Systems, Inc., Bethesda, Md.) is used to detecthuman monoclonal antibody or mouse antibody in the ELISA.

The dose response effect of the monoclonal antibodies are determined forthe antibodies as the lowest antibody titer which reduces virus titersmore than 2 log₁₀ or >99% reduction in virus titer. The degree ofprotection are correlated to the serum antibody levels achieved at thetime of sacrifice. In addition, the potential synergistic effect ofvarious combinations of human monoclonal antibodies is determined. Theresults of the initial in vitro studies outlined above will be used toguide the in vivo experiments. For instance, if RF-1 and RF-2 havedistinct antigenic binding sites on the RSV F protein, combinations ofthe same isotype (γ1 or γ4) may provide for synergistic protection.

ii. Histological evaluation of lung tissue.

The effect of passive therapy on lung inflammation is evaluated bystandard histopathological and immunohistochemical techniques. Bothpenbronchiolar infiltrates and alveolar infiltrates have been describedin the mouse following either primary or secondary infections, Connerset al, J. Virology, (1992), 66, 7444-7451. Experimental animals aretreated with monoclonal antibody, as described above. Uninfecteduntreated control mice serve as comparisons for evaluating histologicaleffects. On days 5 and 8 after infection, the lungs are removed andinflated with formalin under constant filling pressure (30 cm H_(2O))for 30 minutes. After sectioning and staining with hematoxylin-eosin,the degree of inflammatory infiltrate (PMN and lymphocytic separately)in the peribronchiolar and alveolar areas is determined using astandardized scoring system. Since it is anticipated that the γ1 and γ4monoclonal antibodies may fix and activate complement differentially,the lung sections are stained for mouse C3 deposition in areas ofinflammation using a commercially available rabbit anti-mouse C3antibody (Viron Systems, Inc. Bethesda, Md.) and peroxidase conjugatedgoat and-rabbit IgG.

In addition to evaluation of histological changes seen in fixedpulmonary dssues, pulmonary inflammation is assessed by evaluation ofalveolar cytology. Groups of mice, treated as described above, aresacrificed and a bronchoalveolar lavage (BAL) performed by repeatedlyinfusing 3 ml PBS into the lower airway. Cell counts of the BAL wll beperformed, and the cell type identified by staining of cytocentrgepreparations.

iii. Effect of antibody therapy on the nataral immune response toinfection.

In the cotton rat and owl monkey models, passive therapy of RSVinfection with polyclonal IgG preparations minjes the subsequent naturalantibody response to the virus, although animals are failly protectedupon re challenge, Hemming et al., J. Infectous Diseases (1985), 152,1083-1086; Prince et al., Virus Research (1985), 3, 193-206. Incontrast, Graham found that treated mice had both blunted antibodyresponses and were susceptible to virus re challenge, Graham et al.,Ped. Research (1993), 34, 167-172. To assess this possibility usinghuman monoclonal antibodies, mice are infected with the Long strain ofRSV and treated with a protective dose of antibody on day 4, as outlinedabove. Controls will include infected untreated animals, and uninfectedtreated animals. Mice are bled for antibody determination every otherweek for 8 weeks and then every 4 weeks for an additional 8 weeks. Bothhuman monoclonal antibody and mouse antibody to the RSV F and G proteinsare determined by ELISA. In addition, the neutralization titer of theserum determined at each time point. The contributions to neutralizationby the residual human monoclonal antibody and actively produced mouseantiody are inferred from the ELISA results and by the results ofneutralizing activity of the uninfected antibody treated controls. WhenhOman monoclonal antibody is undetectable by ELISA, animals arerechallenged with the same strain of RSV. After 4 days, the animals aresacrificed and the lungs and nasal tissues titered for virus andcompared to control groups.

In order to assess the impact of monoclonal antibody therapy oncytotoxic T-cells (CTL) induction, similar experiments are carried, butsix weeks after infection, mice are sacrificed and spleen cell culturesstimulated with live RSV for 5 days, Walsh, E. E., J. InfectiousDiseases (1994), 170, 345-350. CTL activity is assessed by standardChromium 51 release assay using persistently infected Balb/c fibroblastcell line (BCH4 cells) and compared to an uninfected Balb/c fibroblastline.

Based primarily on the effective dose studies of passive therapy ofestablished RSV infections, it is then determined which antibody, RF-1or RF-2, is the most efficacious for preventing or treating RSVinfection. The choice between the γ1 or γ4 (PE) versions takes the lunghistology studies into account, in particular whether recruitment ofcomplement appear to be significantly enhanced with the Clq bindingantibody. Massive activation of complement could potentially haveadverse effects, although enhanced vascularization that follows mightincrease the virus-antibody confrontation.

c. Test performance in monkey model.

The decisive test for the selected antibody is in a primate model. Wehave chosen the African green monkey (Cercopithecus aethiops) because itis highly permissive for RSV, and infection leads to enhanced lungpathology and detectable lesions, Kakuk et al., J. Infectious Diseases(1993), 167, 553-561. African green monkeys are also readily availableand not endangered. This monkey weighs between 5 and 10 kgs. The highestexpected maximal dose of antibody is 20 mgs/kg. Three animals of each 10kgs with 20 mgs/kg equ 600 mgs of antibody. Some wild African greenmonkeys are naturally immune to RSV, and a requirement for enteringmonkeys into our study is that they are serum negative to RSV.

Based on the baseline established in the mouse model, effective dose/kgand infection time prior to therapy, a limited series of tests areperformed in order to establish effective dose for virus reduction, aswell as to confirm whether this correlates with prevention of lungpathology, in particular parenchymal inflammatory involvement. Only onevirus strain, Long (subtype A) is tested. Initially 25, 5, 1, and 1/25times the reference dose is tested. Two control groups, one that receivevirus but no antibody, and another that receives virus and maximal doseof the isotype matched control antibody, are analyzed. Essentially, theexperiments are effected as described above. Monkeys in groups of 3 arealso infected by intranasal instillation with 10⁶ PFU of virus. Six toseven days after infection with virus the monkeys are sacrificed andlung and pharynx samples are taken for viral assays as described aboveand for histology.

Histology are performed essentially, as described above. Briefly, thelungs are perfused with 10% neutral buffered formalin under constantfilling pressure. The lungs will remain in formalin for at least oneweek. After sectioning and staining with hematoxylin-eosin, the slidesare evaluated histopathologically according to Kakuk et al., J.Infectious Diseases (1993), 167, 553-561. Serum samples are also betaken in order to determine the titer of human are antibody to RSV inELISA and in Infection Neutralization assays.

EXAMPLE 10

1. Confirm tissue specificity by in vitro test on human tissue sections.

The antibody is then further tested for potential cross reactivity tonormal tissues by immunohistology studies on different frozen normaltissue sections from two different individuals. Briefly, Cryostatmicrotome cuts of frozen tissues are subjected to 3 tests: Fixationanalysis, a Nitration analysis and a specificity/distribution analysisPurified biotin labeled anti-RSV F-protein antibody in PBS with 1% BSAis added, and the slide is incubated for 30 min. in a humidified chamberat 200C. The slide is then washed in PBS with I% BSA. The slide issubsequently incubated with Avidin-HRP in PBS with 1% BSA for 30 min.HRP is allowed to react with 3,3 diaminobenzidine-tetrahydrochloride,which forms an insoluble precipitate stain mediated by oxidation withHRP. This will identify any potential cross reactions of the subjecthuman monoclonal antibodies. This test will be performed by ImpathLaboratories, N.Y., N.Y., and is approved by the FDA for I.N.D.submissions for products destined for human therpy. This histologyapproach uses pre-existing tissue and is less costly than thealternative, targeting studies of RSV infected monkeys with radiolabeledantibody.

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(2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1    #/note= "Nucleotide 1 wherein N =              (AG)10."    #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ    #           30     GAAA TTGTGTTGAC    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1    #/note= "Nucleotide 1 wherein N =              (AG)10."    #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ    #           30     GACA TCGTGATGAC    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 31 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1    #/note= "Nucleotide 1 wherein N =              (AG)10."    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 28    #/note= "Nucleotide 28 wherein N =              (G/C)."    #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ    #          31      TTTG ATTTCCANCT T    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 321 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..321    #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ    - GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CT - #G TCT GCA TCT GTC GGA      48    Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly    #                 15    - GAC AGA GTC ACC ATC ACT TGC CGG GCA GGT CA - #G AGG ATT GCT AGT TAT      96    Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Gl - #n Arg Ile Ala Ser Tyr    #             30    - TTA AAT TGG TAT CAG CAC AAA CCA GGG AAA GC - #C CCT AAG CTC CTG ATA     144    Leu Asn Trp Tyr Gln His Lys Pro Gly Lys Al - #a Pro Lys Leu Leu Ile    #         45    - TAT GCT GGA TCC AAT TTG CAC CGT GGG GTC CC - #G TCA AGG TTC AGT GGC     192    Tyr Ala Gly Ser Asn Leu His Arg Gly Val Pr - #o Ser Arg Phe Ser Gly    #     60    - GGT GGA TCT GGG ACA GAT TTC ACT CTC ACC AT - #C AAC AGT CTG CAA CCT     240    Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Il - #e Asn Ser Leu Gln Pro    # 80    - GAA GAT TTT GCA ACT TAC TAT TGT CAA CAG GC - #T TAC AGT ACC CCC TGG     288    Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Al - #a Tyr Ser Thr Pro Trp    #                 95    #        321C CCA GGG ACC AAG GTG GAA ATC AA - #A    Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Ly - #s    #           105    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 378 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..378    #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ    - CAG GTG CAG TTG CAG GAG TCT GGT CCT GTG GT - #G GTG AAA CCC ACA GAG      48    Gln Val Gln Leu Gln Glu Ser Gly Pro Val Va - #l Val Lys Pro Thr Glu    #       120    - ACC CTC ACG CTG ACC TGC ACC GTC TCT GGG TT - #C TCA CTC AGC AAC CCT      96    Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Ph - #e Ser Leu Ser Asn Pro    #   135    - AGA ATG GGT GTG ACC TGG ATC CGT CAG CCC CC - #C GGG AAG GCC CTA GAA     144    Arg Met Gly Val Thr Trp Ile Arg Gln Pro Pr - #o Gly Lys Ala Leu Glu    140                 1 - #45                 1 - #50                 1 -    #55    - TGG CTT GGA AAC ATT TTT TCG AGT GAC GAG AA - #G TCC TTC AGT CCT TCT     192    Trp Leu Gly Asn Ile Phe Ser Ser Asp Glu Ly - #s Ser Phe Ser Pro Ser    #               170    - CTG AAG AGC AGA CTC ACC ACC TCC CAG GAC AC - #C TCC AGA AGC CAG GTG     240    Leu Lys Ser Arg Leu Thr Thr Ser Gln Asp Th - #r Ser Arg Ser Gln Val    #           185    - GTC CTA AGC TTG ACC AAC GTG GAC CCT GTG GA - #C ACA GCC ACA TAT TAC     288    Val Leu Ser Leu Thr Asn Val Asp Pro Val As - #p Thr Ala Thr Tyr Tyr    #       200    - TGT GCA CGG GTA GGA CTG TAT GAC ATC AAT GC - #T TAT TAC CTA TAC TAC     336    Cys Ala Arg Val Gly Leu Tyr Asp Ile Asn Al - #a Tyr Tyr Leu Tyr Tyr    #   215    - CTG GAT TAT TGG GGG CAG GGA ACC CTG GTC AC - #C GTC TCC TCA    # 378    Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Th - #r Val Ser Ser    220                 2 - #25                 2 - #30    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 318 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..318    #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ    - GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CT - #G TCT GCA TCT GTC GGA      48    Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly    #           140    - GAC AGA GTC ACC ATC ACT TGC CGG GCA AGT CA - #G AGC ATT GCC AGT TAT      96    Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gl - #n Ser Ile Ala Ser Tyr    #       155    - GTA AAT TGG TAT CAA CAG AAA CCA GGG AAA GC - #C CCT AAA GTC CTC ATT     144    Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Al - #a Pro Lys Val Leu Ile    #   170    - TTT GCT TCA GCC AAT TTG GTG AGT GGG GTC CC - #A TCA AGA TTC AGT GGC     192    Phe Ala Ser Ala Asn Leu Val Ser Gly Val Pr - #o Ser Arg Phe Ser Gly    175                 1 - #80                 1 - #85                 1 -    #90    - AGT GGA TCT GGG ACA GTT TTC ACC CTC ACC AT - #C AGC AAT CTG CAA CCT     240    Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Il - #e Ser Asn Leu Gln Pro    #               205    - GAA GAT TTT GCA ACC TAC TTC TGT CAG CAG AG - #T TAC ACT AAT TTC AGT     288    Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Se - #r Tyr Thr Asn Phe Ser    #           220    #          318     CC AAG CTG GAA ATC AAA    Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys    #       230    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 378 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..378    #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ    - CAG GTA CAG TTG CAG GAG TCT GGT CCT GCG CT - #G GTA AAA CCC ACA CAG      48    Gln Val Gln Leu Gln Glu Ser Gly Pro Ala Le - #u Val Lys Pro Thr Gln    #                 15    - ACC CTC ACA CTG ACC TGC ACC TTC TCT GGG TT - #C TCA CTC AGC ACC AGA      96    Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Ph - #e Ser Leu Ser Thr Arg    #             30    - GGA ATG AGT GTG AAC TGG ATC CGT CAG CCC CC - #A GGG AAG GCC CTG GAA     144    Gly Met Ser Val Asn Trp Ile Arg Gln Pro Pr - #o Gly Lys Ala Leu Glu    #         45    - TGG CTA GCC CGC ATT GAT TGG GAC GAT GAT AC - #A TTC TAC AGC GCT TCT     192    Trp Leu Ala Arg Ile Asp Trp Asp Asp Asp Th - #r Phe Tyr Ser Ala Ser    #     60    - CTG AAG ACT AGG CTC AGC ATC TCC AAG GAC AC - #C TCC AAA AAC CAG GTG     240    Leu Lys Thr Arg Leu Ser Ile Ser Lys Asp Th - #r Ser Lys Asn Gln Val    # 80    - GTC CTC AGA ATG ACC AAC GTA GAC CCT GTG GA - #C ACA GCC ACA TAT TTT     288    Val Leu Arg Met Thr Asn Val Asp Pro Val As - #p Thr Ala Thr Tyr Phe    #                 95    - TGT GCA CGG GCC TCA CTA TAT GAC AGT GAT AG - #T TTC TAC CTC TTC TAC     336    Cys Ala Arg Ala Ser Leu Tyr Asp Ser Asp Se - #r Phe Tyr Leu Phe Tyr    #           110    - CAT GCC TAC TGG GGC CAG GGA ACC GTG GTC AC - #C GTC TCC TCA    # 378    His Ala Tyr Trp Gly Gln Gly Thr Val Val Th - #r Val Ser Ser    #       125    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 705 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..705    #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ    - ATG GAG ACC CCT GCT CAG CTC CTG GGG CTC CT - #G CTA CTC TGG CTC CGA      48    Met Glu Thr Pro Ala Gln Leu Leu Gly Leu Le - #u Leu Leu Trp Leu Arg    #           140    - GGT GCC AGA TGT GAC ATC CAG ATG ACC CAG TC - #T CCA TCC TCC CTG TCT      96    Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Se - #r Pro Ser Ser Leu Ser    #       155    - GCA TCT GTC GGA GAC AGA GTC ACC ATC ACT TG - #C CGG GCA GGT CAG AGG     144    Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cy - #s Arg Ala Gly Gln Arg    #   170    - ATT GCT AGT TAT TTA AAT TGG TAT CAG CAC AA - #A CCA GGG AAA GCC CCT     192    Ile Ala Ser Tyr Leu Asn Trp Tyr Gln His Ly - #s Pro Gly Lys Ala Pro    175                 1 - #80                 1 - #85                 1 -    #90    - AAG CTC CTG ATA TAT GCT GGA TCC AAT TTG CA - #C CGT GGG GTC CCG TCA     240    Lys Leu Leu Ile Tyr Ala Gly Ser Asn Leu Hi - #s Arg Gly Val Pro Ser    #               205    - AGG TTC AGT GGC GGT GGA TCT GGG ACA GAT TT - #C ACT CTC ACC ATC AAC     288    Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Ph - #e Thr Leu Thr Ile Asn    #           220    - AGT CTG CAA CCT GAA GAT TTT GCA ACT TAC TA - #T TGT CAA CAG GCT TAC     336    Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Ty - #r Cys Gln Gln Ala Tyr    #       235    - AGT ACC CCC TGG ACT TTC GGC CCA GGG ACC AA - #G GTG GAA ATC AAA CGT     384    Ser Thr Pro Trp Thr Phe Gly Pro Gly Thr Ly - #s Val Glu Ile Lys Arg    #   250    - ACG GTG GCT GCA CCA TCT GTC TTC ATC TTC CC - #G CCA TCT GAT GAG CAG     432    Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pr - #o Pro Ser Asp Glu Gln    255                 2 - #60                 2 - #65                 2 -    #70    - TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC CT - #G CTG AAT AAC TTC TAT     480    Leu Lys Ser Gly Thr Ala Ser Val Val Cys Le - #u Leu Asn Asn Phe Tyr    #               285    - CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GA - #T AAC GCC CTC CAA TCG     528    Pro Arg Glu Ala Lys Val Gln Trp Lys Val As - #p Asn Ala Leu Gln Ser    #           300    - GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GA - #C AGC AAG GAC AGC ACC     576    Gly Asn Ser Gln Glu Ser Val Thr Glu Gln As - #p Ser Lys Asp Ser Thr    #       315    - TAC AGC CTC AGC AGC ACC CTG ACG CTG AGC AA - #A GCA GAC TAC GAG AAA     624    Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Ly - #s Ala Asp Tyr Glu Lys    #   330    - CAC AAA GTC TAC GCC TGC GAA GTC ACC CAT CA - #G GGC CTG AGC TCG CCC     672    His Lys Val Tyr Ala Cys Glu Val Thr His Gl - #n Gly Leu Ser Ser Pro    335                 3 - #40                 3 - #45                 3 -    #50    #        705G AGC TTC AAC AGG GGA GAG TGT TG - #A    #*l Thr Lys Ser Phe Asn Arg Gly Glu Cys    #               360    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1428 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..1428    #ID NO:17:(xi) SEQUENCE DESCRIPTION: SEQ    - ATG GGT TGG AGC CTC ATC TTG CTC TTC CTT GT - #C GCT GTT GCT ACG CGT      48    Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Va - #l Ala Val Ala Thr Arg    #               250    - GTC CTG TCC CAG GTG CAG TTG CAG GAG TCT GG - #T CCT GTG GTG GTG AAA      96    Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gl - #y Pro Val Val Val Lys    #           265    - CCC ACA GAG ACC CTC ACG CTG ACC TGC ACC GT - #C TCT GGG TTC TCA CTC     144    Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Va - #l Ser Gly Phe Ser Leu    #       280    - AGC AAC CCT AGA ATG GGT GTG ACC TGG ATC CG - #T CAG CCC CCC GGG AAG     192    Ser Asn Pro Arg Met Gly Val Thr Trp Ile Ar - #g Gln Pro Pro Gly Lys    #   295    - GCC CTA GAA TGG CTT GGA AAC ATT TTT TCG AG - #T GAC GAG AAG TCC TTC     240    Ala Leu Glu Trp Leu Gly Asn Ile Phe Ser Se - #r Asp Glu Lys Ser Phe    300                 3 - #05                 3 - #10                 3 -    #15    - AGT CCT TCT CTG AAG AGC AGA CTC ACC ACC TC - #C CAG GAC ACC TCC AGA     288    Ser Pro Ser Leu Lys Ser Arg Leu Thr Thr Se - #r Gln Asp Thr Ser Arg    #               330    - AGC CAG GTG GTC CTA AGC TTG ACC AAC GTG GA - #C CCT GTG GAC ACA GCC     336    Ser Gln Val Val Leu Ser Leu Thr Asn Val As - #p Pro Val Asp Thr Ala    #           345    - ACA TAT TAC TGT GCA CGG GTA GGA CTG TAT GA - #C ATC AAT GCT TAT TAC     384    Thr Tyr Tyr Cys Ala Arg Val Gly Leu Tyr As - #p Ile Asn Ala Tyr Tyr    #       360    - CTA TAC TAC CTG GAT TAT TGG GGG CAG GGA AC - #C CTG GTC ACC GTC TCC     432    Leu Tyr Tyr Leu Asp Tyr Trp Gly Gln Gly Th - #r Leu Val Thr Val Ser    #   375    - TCA GCT AGC ACC AAG GGC CCA TCG GTC TTC CC - #C CTG GCA CCC TCC TCC     480    Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pr - #o Leu Ala Pro Ser Ser    380                 3 - #85                 3 - #90                 3 -    #95    - AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GG - #C TGC CTG GTC AAG GAC     528    Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gl - #y Cys Leu Val Lys Asp    #               410    - TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AA - #C TCA GGC GCC CTG ACC     576    Tyr Phe Pro Glu Pro Val Thr Val Ser Trp As - #n Ser Gly Ala Leu Thr    #           425    - AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CA - #G TCC TCA GGA CTC TAC     624    Ser Gly Val His Thr Phe Pro Ala Val Leu Gl - #n Ser Ser Gly Leu Tyr    #       440    - TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AG - #C AGC TTG GGC ACC CAG     672    Ser Leu Ser Ser Val Val Thr Val Pro Ser Se - #r Ser Leu Gly Thr Gln    #   455    - ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AG - #C AAC ACC AAG GTG GAC     720    Thr Tyr Ile Cys Asn Val Asn His Lys Pro Se - #r Asn Thr Lys Val Asp    460                 4 - #65                 4 - #70                 4 -    #75    - AAG AAA GCA GAG CCC AAA TCT TGT GAC AAA AC - #T CAC ACA TGC CCA CCG     768    Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Th - #r His Thr Cys Pro Pro    #               490    - TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TC - #A GTC TTC CTC TTC CCC     816    Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Se - #r Val Phe Leu Phe Pro    #           505    - CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CG - #G ACC CCT GAG GTC ACA     864    Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Ar - #g Thr Pro Glu Val Thr    #       520    - TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC CC - #T GAG GTC AAG TTC AAC     912    Cys Val Val Val Asp Val Ser His Glu Asp Pr - #o Glu Val Lys Phe Asn    #   535    - TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GC - #C AAG ACA AAG CCG CGG     960    Trp Tyr Val Asp Gly Val Glu Val His Asn Al - #a Lys Thr Lys Pro Arg    540                 5 - #45                 5 - #50                 5 -    #55    - GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GT - #C AGC GTC CTC ACC GTC    1008    Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Va - #l Ser Val Leu Thr Val    #               570    - CTG CAC CAG GAG TGG CTG AAT GGC AAG GAG TA - #C AAG TGC AAG GTC TCC    1056    Leu His Gln Glu Trp Leu Asn Gly Lys Glu Ty - #r Lys Cys Lys Val Ser    #           585    - AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA AC - #C ATC TCC AAA GCC AAA    1104    Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Th - #r Ile Ser Lys Ala Lys    #       600    - GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CT - #G CCC CCA TCC CGG GAT    1152    Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Le - #u Pro Pro Ser Arg Asp    #   615    - GAG CTG ACC AAG AAC CAG GTC AGC CTG ACC TG - #C CTG GTC AAA GGC TTC    1200    Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cy - #s Leu Val Lys Gly Phe    620                 6 - #25                 6 - #30                 6 -    #35    - TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AG - #C AAT GGG CAG CCG GAG    1248    Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Se - #r Asn Gly Gln Pro Glu    #               650    - AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GA - #C TCC GAC GGC TCC TTC    1296    Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu As - #p Ser Asp Gly Ser Phe    #           665    - TTC CTC TAC AGC AAG CTC ACC GTG GAC AAG AG - #C AGG TGG CAG CAG GGG    1344    Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Se - #r Arg Trp Gln Gln Gly    #       680    - AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GC - #T CTG CAC AAC CAC TAC    1392    Asn Val Phe Ser Cys Ser Val Met His Glu Al - #a Leu His Asn His Tyr    #   695    #     1428AAG AGC CTC TCC CTG TCT CCG GGT AA - #A TGA    Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ly - #s  *    700                 7 - #05                 7 - #10    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 708 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..708    #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ    - ATG GAC ATG AGG GTC CCC GCT CAG CTC CTG GG - #G CTC CTG CTA CTC TGG      48    Met Asp Met Arg Val Pro Ala Gln Leu Leu Gl - #y Leu Leu Leu Leu Trp    #           490    - CTC CGA GGT GCC AGA TGT GAC ATC CAG ATG AC - #C CAG TCT CCA TCC TCC      96    Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Th - #r Gln Ser Pro Ser Ser    #       505    - CTG TCT GCA TCT GTC GGA GAC AGA GTC ACC AT - #C ACT TGC CGG GCA AGT     144    Leu Ser Ala Ser Val Gly Asp Arg Val Thr Il - #e Thr Cys Arg Ala Ser    #   520    - CAG AGC ATT GCC AGT TAT GTA AAT TGG TAT CA - #A CAG AAA CCA GGG AAA     192    Gln Ser Ile Ala Ser Tyr Val Asn Trp Tyr Gl - #n Gln Lys Pro Gly Lys    525                 5 - #30                 5 - #35                 5 -    #40    - GCC CCT AAA GTC CTC ATT TTT GCT TCA GCC AA - #T TTG GTG AGT GGG GTC     240    Ala Pro Lys Val Leu Ile Phe Ala Ser Ala As - #n Leu Val Ser Gly Val    #               555    - CCA TCA AGA TTC AGT GGC AGT GGA TCT GGG AC - #A GTT TTC ACC CTC ACC     288    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Th - #r Val Phe Thr Leu Thr    #           570    - ATC AGC AAT CTG CAA CCT GAA GAT TTT GCA AC - #C TAC TTC TGT CAG CAG     336    Ile Ser Asn Leu Gln Pro Glu Asp Phe Ala Th - #r Tyr Phe Cys Gln Gln    #       585    - AGT TAC ACT AAT TTC AGT TTT GGC CAG GGG AC - #C AAG CTG GAA ATC AAA     384    Ser Tyr Thr Asn Phe Ser Phe Gly Gln Gly Th - #r Lys Leu Glu Ile Lys    #   600    - CGT ACG GTG GCT GCA CCA TCT GTC TTC ATC TT - #C CCG CCA TCT GAT GAG     432    Arg Thr Val Ala Ala Pro Ser Val Phe Ile Ph - #e Pro Pro Ser Asp Glu    605                 6 - #10                 6 - #15                 6 -    #20    - CAG TTG AAA TCT GGA ACT GCC TCT GTT GTG TG - #C CTG CTG AAT AAC TTC     480    Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cy - #s Leu Leu Asn Asn Phe    #               635    - TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG GT - #G GAT AAC GCC CTC CAA     528    Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Va - #l Asp Asn Ala Leu Gln    #           650    - TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CA - #G GAC AGC AAG GAC AGC     576    Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gl - #n Asp Ser Lys Asp Ser    #       665    - ACC TAC AGC CTC AGC AGC ACC CTG ACG CTG AG - #C AAA GCA GAC TAC GAG     624    Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Se - #r Lys Ala Asp Tyr Glu    #   680    - AAA CAC AAA GTC TAC GCC TGC GAA GTC ACC CA - #T CAG GGC CTG AGC TCG     672    Lys His Lys Val Tyr Ala Cys Glu Val Thr Hi - #s Gln Gly Leu Ser Ser    685                 6 - #90                 6 - #95                 7 -    #00    #      708ACA AAG AGC TTC AAC AGG GGA GAG TG - #T TGA    Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cy - #s  *    #               710    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1428 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..1428    #ID NO:19:(xi) SEQUENCE DESCRIPTION: SEQ    - ATG GGT TGG AGC CTC ATC TTG CTC TTC CTT GT - #C GCT GTT GCT ACG CGT      48    Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Va - #l Ala Val Ala Thr Arg    #           250    - GTC TTG TCC CAG GTA CAG TTG CAG GAG TCT GG - #T CCT GCG CTG GTA AAA      96    Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gl - #y Pro Ala Leu Val Lys    #       265    - CCC ACA CAG ACC CTC ACA CTG ACC TGC ACC TT - #C TCT GGG TTC TCA CTC     144    Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Ph - #e Ser Gly Phe Ser Leu    #   280    - AGC ACC AGA GGA ATG AGT GTG AAC TGG ATC CG - #T CAG CCC CCA GGG AAG     192    Ser Thr Arg Gly Met Ser Val Asn Trp Ile Ar - #g Gln Pro Pro Gly Lys    285                 2 - #90                 2 - #95                 3 -    #00    - GCC CTG GAA TGG CTA GCC CGC ATT GAT TGG GA - #C GAT GAT ACA TTC TAC     240    Ala Leu Glu Trp Leu Ala Arg Ile Asp Trp As - #p Asp Asp Thr Phe Tyr    #               315    - AGC GCT TCT CTG AAG ACT AGG CTC AGC ATC TC - #C AAG GAC ACC TCC AAA     288    Ser Ala Ser Leu Lys Thr Arg Leu Ser Ile Se - #r Lys Asp Thr Ser Lys    #           330    - AAC CAG GTG GTC CTC AGA ATG ACC AAC GTA GA - #C CCT GTG GAC ACA GCC     336    Asn Gln Val Val Leu Arg Met Thr Asn Val As - #p Pro Val Asp Thr Ala    #       345    - ACA TAT TTT TGT GCA CGG GCC TCA CTA TAT GA - #C AGT GAT AGT TTC TAC     384    Thr Tyr Phe Cys Ala Arg Ala Ser Leu Tyr As - #p Ser Asp Ser Phe Tyr    #   360    - CTC TTC TAC CAT GCC TAC TGG GGC CAG GGA AC - #C GTG GTC ACC GTC TCC     432    Leu Phe Tyr His Ala Tyr Trp Gly Gln Gly Th - #r Val Val Thr Val Ser    365                 3 - #70                 3 - #75                 3 -    #80    - TCA GCT AGC ACC AAG GGC CCA TCG GTC TTC CC - #C CTG GCA CCC TCC TCC     480    Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pr - #o Leu Ala Pro Ser Ser    #               395    - AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GG - #C TGC CTG GTC AAG GAC     528    Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gl - #y Cys Leu Val Lys Asp    #           410    - TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AA - #C TCA GGC GCC CTG ACC     576    Tyr Phe Pro Glu Pro Val Thr Val Ser Trp As - #n Ser Gly Ala Leu Thr    #       425    - AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CA - #G TCC TCA GGA CTC TAC     624    Ser Gly Val His Thr Phe Pro Ala Val Leu Gl - #n Ser Ser Gly Leu Tyr    #   440    - TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AG - #C AGC TTG GGC ACC CAG     672    Ser Leu Ser Ser Val Val Thr Val Pro Ser Se - #r Ser Leu Gly Thr Gln    445                 4 - #50                 4 - #55                 4 -    #60    - ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AG - #C AAC ACC AAG GTG GAC     720    Thr Tyr Ile Cys Asn Val Asn His Lys Pro Se - #r Asn Thr Lys Val Asp    #               475    - AAG AAA GCA GAG CCC AAA TCT TGT GAC AAA AC - #T CAC ACA TGC CCA CCG     768    Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Th - #r His Thr Cys Pro Pro    #           490    - TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TC - #A GTC TTC CTC TTC CCC     816    Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Se - #r Val Phe Leu Phe Pro    #       505    - CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CG - #G ACC CCT GAG GTC ACA     864    Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Ar - #g Thr Pro Glu Val Thr    #   520    - TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC CC - #T GAG GTC AAG TTC AAC     912    Cys Val Val Val Asp Val Ser His Glu Asp Pr - #o Glu Val Lys Phe Asn    525                 5 - #30                 5 - #35                 5 -    #40    - TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GC - #C AAG ACA AAG CCG CGG     960    Trp Tyr Val Asp Gly Val Glu Val His Asn Al - #a Lys Thr Lys Pro Arg    #               555    - GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GT - #C AGC GTC CTC ACC GTC    1008    Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Va - #l Ser Val Leu Thr Val    #           570    - CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TA - #C AAG TGC AAG GTC TCC    1056    Leu His Gln Asp Trp Leu Asn Gly Lys Glu Ty - #r Lys Cys Lys Val Ser    #       585    - AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA AC - #C ATC TCC AAA GCC AAA    1104    Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Th - #r Ile Ser Lys Ala Lys    #   600    - GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CT - #G CCC CCA TCC CGG GAT    1152    Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Le - #u Pro Pro Ser Arg Asp    605                 6 - #10                 6 - #15                 6 -    #20    - GAG CTG ACC AAG AAC CAG GTC AGC CTG ACC TG - #C CTG GTC AAA GGC TTC    1200    Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cy - #s Leu Val Lys Gly Phe    #               635    - TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AG - #C AAT GGG CAG CCG GAG    1248    Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Se - #r Asn Gly Gln Pro Glu    #           650    - AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GA - #C TCC GAC GGC TCC TTC    1296    Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu As - #p Ser Asp Gly Ser Phe    #       665    - TTC CTC TAC AGC AAG CTC ACC GTG GAC AAG AG - #C AGG TGG CAG CAG GGG    1344    Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Se - #r Arg Trp Gln Gln Gly    #   680    - AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GC - #T CTG CAC AAC CAC TAC    1392    Asn Val Phe Ser Cys Ser Val Met His Glu Al - #a Leu His Asn His Tyr    685                 6 - #90                 6 - #95                 7 -    #00    #     1428AAG AGC CTC TCC CTG TCT CCG GGT AA - #A TGA    Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ly - #s  *    #               710    __________________________________________________________________________

We claim:
 1. Eukaryotic cells which have been transfected with DNAsequences which encode for the heavy and light variable domains ofanti-RSV (Respiratory Syncytial Virus) F-protein human monoclonalantibody either RF-1 or RF-2.
 2. The cells of claim 1 wherein saideukaryotic cells are CHO cells.
 3. The eukaryotic cells of claim 1wherein said DNA sequences are selected from the DNA sequences set forthin any one of FIGS. 7a, 7b, 8a, 8b, 9a, 9b, 11a and 11b.
 4. AnEpstein-Barr immortalized B cell line which secretes a human monoclonalantibody which possesses an affinity (Kd) for the RSV respiratorySyncytial virus fusion protein of about 2×10⁻⁹ to 10⁻¹⁰ molar.
 5. Thecell line of claim 4 wherein said antibody neutralizes RSV in vitro. 6.The cell line of claim 4 wherein said cell line is selected from thegroup consisting of RF-1 and RF-2.
 7. A DNA sequence which encodes forthe variable heavy and/or variable light domain of anti-RSV (RespiratorySyncytial Virus) F-protein human monoclonal antibody RF-1 or RF-2.
 8. Anexpression vector which provides for the expression of a DNA sequenceaccording to claim
 7. 9. The DNA sequence of claim 7 which is selectedfrom the group consisting of the DNA sequences set forth in FIGS. 7a,7b, 8a, 8b, 9a, 9b, 10a and 10b.